Personalized cells, tissues, and organs for transplantation from a humanized, bespoke, designated-pathogen free, (non-human) donor and methods and products relating to same

ABSTRACT

A biological system for generating and preserving a repository of personalized, humanized transplantable cells, tissues, and organs for transplantation, wherein the biological system is biologically active and metabolically active, the biological system having genetically reprogrammed cells, tissues, and organs in a non-human animal for transplantation into a human recipient, wherein the non-human animal does not present one or more surface glycan epitopes and specific sequences from the wild-type swine&#39;s SLA is replaced with a synthetic nucleotides based on a human captured reference sequence from a human recipient&#39;s HLA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. provisional patent applicationNo. 62/975,611, filed Feb. 12, 2020, U.S. provisional patent applicationNo. 62/964,397, filed Jan. 22, 2020, U.S. provisional patent applicationNo. 62/848,272, filed May 15, 2019, U.S. provisional patent applicationNo. 62/823,455, filed Mar. 25, 2019, and U.S. non-provisional patentapplication Ser. No. 16/59378, filed Oct. 4, 2019, which claims prioritybenefit of U.S. provisional application No. 62/742,188, filed Oct. 5,2018; 62/756,925, filed Nov. 7, 2018; U.S. 62/756,955 filed Nov. 7,2018; U.S. 62/756,977, filed Nov. 7, 2018; U.S. 62/756,993, filed Nov.7, 2018; U.S. 62/792,282, filed Jan. 14, 2019; U.S. 62/795,527, filedJan. 22, 2019; U.S. 62/823,455, filed Mar. 25, 2019; and U.S.62/848,272, filed May 15, 2019, the disclosures of all of which areincorporated herein by reference in their entireties. The instantapplication contains a Sequence Listing which has been submitted on thefiling date of Mar. 25, 2020 via EFS-Web and is incorporated byreference in its entirety. Said Sequence Listing, created on Mar. 25,2020, is named 4772-108_ST25.TXT and is 87 kilobytes in size.

BACKGROUND OF THE INVENTION

According to the United Network for Organ Sharing (“UNOS”), every tenminutes, someone is added to the national transplant waiting list, andnearly 20 people die each day waiting for a transplant. As of March2020, there were about 112,385 people in need of a lifesaving organtransplant in the United States, with only about 19,000 donorsidentified and about 39,000 transplants performed in 2019 (data from theUnited Network for Organ Sharing (UNOS)). The need for specific organsin the United States is as follows:

Organ Candidates in Need Kidney 102,730 Liver 12,926 Pancreas 879Kidney/Pancreas 1,820 Heart 3,702 Lung 1,283 Heart/Lung 52 Intestine 238Total 124,630

Over the past 5 years, from 2014 through 2019, an average of about 6,400candidates died each year while on the waiting list and withoutreceiving an organ transplant. About the same number were not able toreceive a long-awaited transplant because they were too sick to receivea transplant for the requisite surgery. While the rate of divergencebetween available donors and unmet need of recipients has been improvedmarginally, this disparity has continued to present day and remainsconsiderable; the supply remains disastrously inadequate. Of course, thepatients in need are awaiting for organs from human donors, which wouldrepresent the transplantation of organs from one species to another(allotransplantation).

Allotransplantation presents many significant multifaceted problems,involving safety, logistical, ethical, legal, institutional, andcultural complications. From a safety perspective, allogeneic tissuesfrom human donors carry significant infectious disease risks. Forexample some in the transplantation field have report that “[human]cytomegalovirus (CMV) is the single most important infectious agentaffecting recipients of organ transplants, with at least two-thirds ofthese patients having CMV infection after transplantation.” Denner J(2018) Reduction of the survival time of pig xenotransplants by porcinecytomegalovirus. Virology Journal, 15(1): 171; Rubin R H (1990) Impactof cytomegalovirus infection on organ transplant recipients. Reviews ofInfectious Diseases, 12 Suppl 7:S754-766.

Regulations regarding tissue transplants include criteria for donorscreening and testing for adventitious agents, as well as strictregulations that govern the processing and distribution of tissuegrafts. The transmission of viruses has occurred as a result ofallotransplantation. Exogenous retroviruses (Human T-cell leukemia virustype 1 (HTLV-1), Human T-cell leukemia virus type 2 (HTLV-2), and Humanimmunodeficiency virus (HIV) have been transmitted by human tissuesduring organ and cell transplantation, as have viruses such as humancytomegalovirus, and even rabies. Due to technical and timingconstraints surrounding organ viability and post-mortem screening,absolute testing is hindered, and this risk cannot be eliminated.

Immunological disparities between recipient and donor preventgraft-survival for extended durations, without immunosuppressiveregimens that pose their own set of complications and additional risks.When a patient receives an organ from a (non-self) donor (living ordeceased), the recipient's immune system will recognize the transplantas foreign. This recognition will cause their immune system to mobilizeand “reject” the organ unless concomitant medications that suppress theimmune system's natural processes are utilized. The response to anallogeneic skin graft is a potent immune response involving engagementof both the innate and adaptive immune systems. Abbas A K, Lichtman A HH, Pillai S (2017) Cellular and Molecular Immunology.

With regard to the use of immunosuppressants, immunosuppressive drugsprolong survival of the transplanted graft in acute and chronicrejection schemas. However, they leave patients vulnerable to infectionsfrom even the most routine of pathogens and require continued use forlife but expose the patient to an increased risk of infection, evencancer. immunosuppressant can blunt the natural immunological processes;unfortunately, these medications are often a lifelong requirement afterorgan transplantation and increase recipient susceptibility to otherwiseroutine pathogens. While these drugs allow transplant recipients totolerate the presence of foreign organs, they also increase the risk ofinfectious disease and symptoms associated with a compromised immunesystem, as a broad array of organisms may be transmitted with humanallografts.” Fishman J A, Greenwald M A, Grossi P A (2012) Transmissionof Infection With Human Allografts: Essential Considerations in DonorScreening. Clinical Infectious Diseases, 55(5):720-727.

Logistically, numerous factors must be considered prior to a successfulorgan donation and transplant procedure. Blood type and other medicalfactors must be evaluated for every donated organ, but further, eachorgan type presents unique characteristics that also must be weighed,such as post-mortem ischemia, immunological compatibility, patientlocation, and institutional capabilities.

For these patients, and the millions not included in these statisticswho also would benefit significantly from tissue transplants such ascornea or pancreatic islet cells, some in the field have confirmed that“allotransplantation will never prove to be a sufficient source.” EkserB, Cooper D K C, Tector A J (2015) The Need for Xenotransplantation as aSource of Organs and Cells for Clinical Transplantation. Internationaljournal of surgery (London, England), 23(0 0): 199-204.

Despite such drawbacks, organ transplantation is unquestionably thepreferred therapy for most patients with end stage organ failure, inlarge part due to a lack of viable alternatives. However, the advent oforgan transplantation as a successful life-saving therapeuticintervention, juxtaposed against the paucity of organs available totransplant, unfortunately places medical professionals in anideologically vexing position of having to decide who lives and whodies. Ultimately, alternative and adjunct treatment options that wouldminimize the severe shortcomings of allotransplant materials whileproviding the same mechanism of action that makes them so effectivewould be of enormous benefit to patients worldwide.

The urgent need for organs and other transplantation tissue generally,including for temporary therapies while more permanent organs or othertissue are located and utilized, has led to investigation intoutilization of organs, cells and tissue from non-human sources,including other animals for temporary and/or permanentxenotransplantation.

Xenotransplantation, such as the transplantation of a non-human animalorgan into a human recipient, has the potential to reduce the shortageof organs available for transplant, potentially helping thousands ofpeople worldwide. Swine have been considered a potential non-humansource of organs, tissue and/or cells for use in humanxenotransplantation given that their size and physiology are compatiblewith humans. However, xenotransplantation using standard, unmodified pigtissue into a human or other primate is accompanied by rejection of thetransplanted tissue.

Wild type swine organs would evoke rejection by the human immune systemupon transplantation into a human where natural human antibodies targetepitopes on the swine cells, causing rejection and failure of thetransplanted organs, cells or tissue. The rejection may be a cellularrejection (lymphocyte mediated) or humoral (antibody mediated) rejectionincluding but not limited to hyperacute rejection, an acute rejection, achronic rejection, may involve survival limiting thrombocytopeniacoagulopathy and an acute humoral xenograft reaction (AHXR). Otherroadblocks with respect to swine to human xenotransplantation includerisks of cross-species transmission of disease or parasites.

One cause of hyperacute rejection results from the expression ofalpha-1,3-galactosyltransferase (“alpha-1,3-GT”) in porcine cells, whichcauses the synthesis of alpha-1,3-galactose epitopes. Except for humans,apes and Old World monkeys, most mammals carry glycoproteins on theircell surfaces that contain galactose alpha 1,3-galactose (see, e.g.,Galili et al., “Man, apes, and old world monkeys differ from othermammals in the expression of α-galactosyl epitopes on nucleated cells,”J. Biol. Chem. 263: 17755-17762 (1988). Humans, apes and Old Worldmonkeys have a naturally occurring anti-alpha gal antibody that isproduced and binds to glycoproteins and glycolipids having galactosealpha-1,3 galactose (see, e.g., Cooper et al., “Genetically engineeredpigs,” Lancet 342:682-683 (1993)).

Accordingly, when natural type swine products are utilized inxenotransplantation, human antibodies will be invoked to confront theforeign alpha-1,3-galactose epitopes, and hyperacute rejection normallyfollows. Beyond alpha-1,3-GT, swine cells express multiple genes whichare not found in human cells. These include, but are not limited to,Neu5GC, and β1,4-N-acetylgalactosaminyltransferase (B4GALNT2).Antibodies to the α-Gal, Neu5GC, β1,4-N and Sda-like antigens arepresent in human blood prior to implantation of xeno-tissue, and areinvolved in the intense and immediate antibody-mediated rejection ofimplanted tissue.

Additionally pig cells express Class I and Class II SLAs on endothelialcells. The SLA cross-reacting antibodies contribute to the intense andimmediate rejection of the implanted porcine tissue. SLA antigens mayalso be involved with the recipient's T-cell mediated immune response.Porcine SLAs may include, but are not limited to, antigens encoded bythe SLA-1, SLA-2, SLA-3, SLA-4, SLA-5, SLA-6, SLA-8, SLA-9, SLA-11 andSLA-12 loci. Porcine Class II SLAs include antigens encoded by theSLA-DQ and SLA-DR loci.

Many attempts have been made by others to modify swine to serve as asource for xenotransplantation products, however such attempts have notyielded a successful swine model to date. Such commercial, academic andother groups have focused on interventions, gene alterations, efforts toinduce tolerance through chimerism, inclusion of transgenes, concomitantuse of exogenous immunosuppressive medications aimed to reduce therecipients' natural immunologic response(s) and other approaches. Thesegroups have sought to create a “one size fits all” source animal aimingto create one, standardized source animal for all recipients.

Specifically, certain groups have focused on creating transgenic swinefree of PERV and utilizing transgenic bone marrow for therapy (see,e.g., eGenesis, Inc. PCT/US2018/028539); creating transgenic swineutilizing stem cell scaffolding (see, e.g. United Therapeutics/Revivicor[US20190111180A1]); mixed chimerism and utilizing transgenic bone marrowfor therapy to tolerize patient T-cells (see, e.g. Columbia University[US20180070564A1]). These “downstream” approaches—post recognition bythe human immune system—have not succeeded in producing swine thatproduce products suitable for prolonged use in xenotransplantation orthat survive the above-referenced transgenic and other alterations.

In contrast to the above-referenced approaches, the present inventionachieves a “patient-specific” solution by modifying the genome of donorswine cells to escape detection from the human immune system in thefirst instance, avoiding the immune cascade that follows when apatient's T-cells and antibodies are primed to destroy foreign material.This “upstream” approach is achieved through, in one aspect, specificcombinations of minimal genetic alterations that render the donoranimal's cells, tissues, and organs tolerogenic when transplanted into ahuman without sacrificing the animal's immune function. The presentinvention therefore addresses long-felt but unmet need for translatingthe science of xenotransplantation into a clinical reality.

This “upstream” approach is achieved through, in one aspect, specificcombinations of minimal genetic alterations that render the donoranimal's cells, tissues, and organs tolerogenic when transplanted into ahuman without sacrificing the animal's immune function. The presentinvention therefore addresses long-felt but unmet need for translatingthe science of xenotransplantation into a clinical reality.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure includes a biological system forgenerating and preserving a repository of personalized, humanizedtransplantable cells, tissues, and organs for transplantation, whereinthe biological system is biologically active and metabolically active,the biological system comprising genetically reprogrammed cells,tissues, and organs in a non-human animal for transplantation into ahuman recipient. For example, the non-human animal is a geneticallyreprogrammed swine for xenotransplantation of cells, tissue, and/or anorgan isolated from the genetically reprogrammed swine, the geneticallyreprogrammed swine comprising a nuclear genome that has beenreprogrammed to replace a plurality of nucleotides in a plurality ofexon regions of a major histocompatibility complex of a wild-type swinewith a plurality of synthesized nucleotides from a human capturedreference sequence. In one aspect, cells of said geneticallyreprogrammed swine do not present one or more surface glycan epitopesselected from alpha-Gal, Neu5Gc, and SD^(a). Further, genes encodingalpha-1,3 galactosyltransferase, cytidinemonophosphate-N-acetylneuraminic acid hydroxylase (CMAH), andβ1,4-N-acetylgalactosaminyltransferase are altered such that thegenetically reprogrammed swine lacks functional expression of surfaceglycan epitopes encoded by those genes. In some aspects, thereprogrammed genome comprises site-directed mutagenic substitutions ofnucleotides at exon regions of: i) at least one of the wild-type swine'sSLA-1, SLA-2, and SLA-3 with nucleotides from an orthologous exon regionof HLA-A, HLA-B, and HLA-C, respectively, of the human capturedreference sequence; and ii) at least one the wild-type swine's SLA-6,SLA-7, and SLA-8 with nucleotides from an orthologous exon region ofHLA-E, HLA-F, and HLA-G, respectively, of the human captured referencesequence; and iii) at least one of the wild-type swine's SLA-DR andSLA-DQ with nucleotides from an orthologous exon region of HLA-DR andHLA-DQ, respectively, of the human captured reference sequence. In someaspects, the reprogrammed genome comprises at least one of A-C:

-   -   A) wherein the reprogrammed swine nuclear genome comprises        site-directed mutagenic substitutions of nucleotides at exon        regions of the wild-type swine's β2-microglobulin with        nucleotides from orthologous exons of a known human        β2-microglobulin from the human captured reference sequence;    -   B) wherein the reprogrammed swine nuclear genome comprises a        polynucleotide that encodes a polypeptide that is a humanized        beta 2 microglobulin (hB2M) polypeptide sequence that is at        least 95% identical to the amino acid sequence of beta 2        microglobulin glycoprotein expressed by the human captured        reference genome;    -   C) wherein the reprogrammed swine nuclear genome has been        reprogrammed such that, at the swine's endogenous        β2-microglobulin locus, the nuclear genome has been reprogrammed        to comprise a nucleotide sequence encoding β2-microglobulin        polypeptide of the human recipient. Further, in some aspects,        the reprogrammed swine nuclear genome has been reprogrammed such        that the genetically reprogrammed swine lacks functional        expression of the wild-type swine's endogenous β2-microglobulin        polypeptides. Further, the reprogramming does not introduce any        frameshifts or frame disruptions.

In other aspects, the present disclosure includes a method of preparinga genetically reprogrammed swine comprising a nuclear genome that lacksfunctional expression of surface glycan epitopes selected fromalpha-Gal, Neu5Gc, and SD^(a) and is genetically reprogrammed to expressa humanized phenotype of a human captured reference sequence comprising:

-   -   a. obtaining a porcine fetal fibroblast cell, a porcine zygote,        a porcine Induced Pluripotent Stem Cells (IPSC), or a porcine        germ-line cell;    -   b. genetically altering said cell in a) to lack functional        alpha-1,3 galactosyltransferase, cytidine        monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and        β1,4-N-acetylgalactosaminyltransferase;    -   c. genetically reprogramming said cell in b) using clustered        regularly interspaced short palindromic repeats (CRISPR)/Cas for        site-directed mutagenic substitutions of nucleotides at exon        regions of: i) at least one of the wild-type swine's SLA-1,        SLA-2, and SLA-3 with nucleotides from an orthologous exon        region of HLA-A, HLA-B, and HLA-C, respectively, of the human        captured reference sequence; and ii) at least one the wild-type        swine's SLA-6, SLA-7, and SLA-8 with nucleotides from an        orthologous exon region of HLA-E, HLA-F, and HLA-G,        respectively, of the human captured reference sequence; and iii)        at least one of the wild-type swine's SLA-DR and SLA-DQ with        nucleotides from an orthologous exon region of HLA-DR and        HLA-DQ, respectively, of the human captured reference sequence,        wherein intron regions of the wild-type swine's genome are not        reprogrammed, and        wherein the reprogrammed genome comprises at least one of A-C:        A) wherein the reprogrammed swine nuclear genome comprises        site-directed mutagenic substitutions of nucleotides at exon        regions of the wild-type swine's β2-microglobulin with        nucleotides from orthologous exons of a known human        β2-microglobulin from the human captured reference sequence;        B) wherein the reprogrammed swine nuclear genome comprises a        polynucleotide that encodes a polypeptide that is a humanized        beta 2 microglobulin (hB2M) polypeptide sequence that is at        least 95% identical to beta 2 microglobulin expressed by the        human captured reference genome;        C) wherein the reprogrammed swine nuclear genome has been        reprogrammed such that the genetically reprogrammed swine lacks        functional expression of the wild-type swine's endogenous        β2-microglobulin polypeptides, wherein the reprogrammed swine        nuclear genome has been reprogrammed such that, at the swine's        endogenous β2-microglobulin locus, the nuclear genome has been        reprogrammed to comprise a nucleotide sequence encoding        β2-microglobulin polypeptide of the human recipient,

wherein said reprogramming does not introduce any frameshifts or framedisruptions,

-   -   d. generating an embryo from the genetically reprogrammed cell        in c); and    -   e. transferring the embryo into a surrogate pig and growing the        transferred embryo in the surrogate pig.

In another aspect, the present disclosure includes a method of producinga donor swine tissue or organ for xenotransplantation, wherein cells ofsaid donor swine tissue or organ are genetically reprogrammed to becharacterized by a recipient-specific surface phenotype comprising:

-   -   a. obtaining a biological sample containing DNA from a        prospective human transplant recipient;    -   b. performing whole genome sequencing of the biological sample        to obtain a human capture reference sequence;    -   c. comparing the human capture reference sequence with the        wild-type genome of the donor swine at loci (i)-(v):        -   (i) exon regions encoding at least one of SLA-1, SLA-2, and            SLA-3;        -   (ii) exon regions encoding at least one of SLA-6, SLA-7, and            SLA-8;        -   (iii) exon regions encoding at least one of SLA-DR and            SLA-DQ;        -   (iv) one or more exons encoding beta 2 microglobulin (B2M);        -   (v) exon regions of SLA-MIC-2 gene and a gene encoding at            least one of PD-L1, CTLA-4, EPCR, TBM, and TFPI,    -   d. creating synthetic donor swine nucleotide sequences of 10 to        350 basepairs in length for one or more of said loci (i)-(v),        wherein said synthetic donor swine nucleotide sequences are at        least 95% identical to the human capture reference sequence at        orthologous loci (vi)-(x) corresponding to swine loci (i)-(vi),        respectively:        -   (vi) exon regions encoding at least one of HLA-A, HLA-B, and            HLA-C;        -   (vii) exon regions encoding at least one of HLA-E, HLA-F,            and HLA-G;        -   (viii) exon regions encoding at least one of HLA-DR and            HLA-DQ;        -   (ix) one or more exons encoding human beta 2 microglobulin            (hB2M);        -   (x) exon regions encoding at least one of MIC-A, MIC-B,            PD-L1, CTLA-4, EPCR, TBM, and TFPI from the human capture            reference sequence,    -   e. replacing nucleotide sequences in (i)-(v) with said synthetic        donor swine nucleotide sequences; and    -   f. obtaining the swine tissue or organ for xenotransplantation        from a genetically reprogrammed swine having said synthetic        donor swine nucleotide sequences.

In another aspect, the present disclosure includes a method of screeningfor off target edits or genome alterations in the geneticallyreprogrammed swine comprising a nuclear genome of the present disclosureincluding:

-   -   a. performing whole genome sequencing on a biological sample        containing DNA from a donor swine before performing genetic        reprogramming of the donor swine nuclear genome, thereby        obtaining a first whole genome sequence;    -   b. after reprogramming of the donor swine nuclear genome,        performing whole genome sequencing to obtain a second whole        genome sequence;    -   c. aligning the first whole genome sequence and the second whole        genome sequence to obtain a sequence alignment;    -   d. analyzing the sequence alignment to identify any mismatches        to the swine's genome at off-target sites.

In another aspect, the present disclosure includes a syntheticnucleotide sequence having wild-type swine intron regions from awild-type swine MHC Class Ia, and reprogrammed at exon regions encodingthe wild-type swine's SLA-3 with codons of HLA-C from a human capturereference sequence that encode amino acids that are not conservedbetween the SLA-3 and the HLA-C from the human capture referencesequence. In some aspects, the wild-type swine's SLA-1 and SLA-2 eachcomprise a stop codon.

In another aspect, the present disclosure includes a syntheticnucleotide sequence having wild-type swine intron regions from awild-type swine MHC Class Ib, and reprogrammed at exon regions encodingthe wild-type swine's SLA-6, SLA-7, and SLA-8 with codons of HLA-E,HLA-F, and HLA-G, respectively, from a human capture reference sequencethat encode amino acids that are not conserved between the SLA-6, SLA-7,and SLA-8 and the HLA-E, HLA-F, and HLA-G, respectively, from the humancapture reference sequence.

In another aspect, the present disclosure includes a syntheticnucleotide sequence having wild-type swine intron regions from awild-type swine MHC Class II, and reprogrammed at exon regions encodingthe wild-type swine's SLA-DQ with codons of HLA-DQ, respectively, from ahuman capture reference sequence that encode amino acids that are notconserved between the SLA-DQ and the HLA-DQ, respectively, from thehuman capture reference sequence, and wherein the wild-type swine'sSLA-DR comprises a stop codon.

In another aspect, the present disclosure includes a syntheticnucleotide sequence having wild-type swine intron regions from awild-type swine beta-2-microglobulin and reprogrammed at exon regionsencoding the wild-type swine's beta-2-microglobulin with codons ofbeta-2-microglobulin from a human capture reference sequence that encodeamino acids that are not conserved between the wild-type swine'sbeta-2-microglobulin and the beta-2-microglobulin from the human capturereference sequence, wherein the synthetic nucleotide sequence comprisesat least one stop codon in an exon region such that the syntheticnucleotide sequence lacks functional expression of the wild-type swine'sβ2-microglobulin polypeptides.

In another aspect, the present disclosure includes a syntheticnucleotide sequence having wild-type swine intron regions from awild-type swine MIC-2, and reprogrammed at exon regions of the wild-typeswine's MIC-2 with codons of MIC-A or MIC-B from a human capturereference sequence that encode amino acids that are not conservedbetween the MIC-2 and the MIC-A or the MIC-B from the human capturereference sequence.

In another aspect, the present disclosure includes a syntheticnucleotide sequence having wild-type swine intron regions from awild-type swine CTLA-4, and reprogrammed at exon regions encoding thewild-type swine's CTLA-4 with codons of CTLA-4 from a human capturereference sequence that encode amino acids that are not conservedbetween the wild-type swine's CTLA-4 and the CTLA-4 from the humancapture reference sequence.

In another aspect, the present disclosure includes a syntheticnucleotide sequence having wild-type swine intron regions from awild-type swine PD-L1 and reprogrammed at exon regions encoding thewild-type swine's PD-L1 with codons of PD-L1 from a human capturereference sequence that encode amino acids that are not conservedbetween the wild-type swine's PD-L1 and the PD-L1 from the human capturereference sequence.

In another aspect, the present disclosure includes a syntheticnucleotide sequence having wild-type swine intron regions from awild-type swine EPCR and reprogrammed at exon regions encoding thewild-type swine's EPCR with codons of EPCR from a human capturereference sequence that encode amino acids that are not conservedbetween the wild-type swine's EPCR and the EPCR from the human capturereference sequence.

In another aspect, the present disclosure includes a syntheticnucleotide sequence having wild-type swine intron regions from awild-type swine TBM and reprogrammed at exon regions encoding thewild-type swine's TBM with codons of TBM from a human capture referencesequence that encode amino acids that are not conserved between thewild-type swine's TBM and the TBM from the human capture referencesequence.

In another aspect, the present disclosure includes a syntheticnucleotide sequence having wild-type swine intron regions from awild-type swine TFPI and reprogrammed at exon regions encoding thewild-type swine's TFPI with codons of TFPI from a human capturereference sequence that encode amino acids that are not conservedbetween the wild-type swine's TFPI and the TFPI from the human capturereference sequence.

In contrast to the above-referenced approaches, the present inventionachieves a “patient-specific” solution by modifying the genome of donorswine cells to escape detection from the human immune system in thefirst instance, avoiding the immune cascade that follows when apatient's T-cells and antibodies are primed to destroy foreign material.This “upstream” approach is achieved through, in one aspect, minimal,modifications to the swine genome involving distinct combinations ofdisruptions (such as knocking out α1,3-galactosyltransferase (αGal),cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) and/orβ1-4 N-acetylgalactosaminyltransferase such that the donor swine cellsdo not express such on its cell surfaces), regulation of expression ofcertain genes (for example, CTLA-4 and PD-1), and replacement ofspecific sections of the swine genome with synthetically engineeredsections based upon recipient human capture sequences (for example, incertain SLA sequences to regulate the swine's expression of, forexample, MHC-I and MHC-II). The present invention therefore addresseslong-felt but unmet need for translating the science ofxenotransplantation into a clinical reality.

Such modifications result in the reduce the extent of, the causative,immunological disparities and associated, deleterious immune processesthat result from the recognition of “non-self”, by selectively alteringthe extracellular antigens of the donor to increase the likelihood ofacceptance of the transplant.

In certain aspects, the present disclosure centralizes (predicates) thecreation of hypoimmunogenic and/or tolerogenic cells, tissues, andorgans that does not necessitate the transplant recipients' prevalentand deleterious use of exogenous immunosuppressive drugs (or prolongedimmunosuppressive regimens) following the transplant procedure toprolong the life-saving graft. This approach is countervailing to theexisting and previous dogmatic approaches; instead of accepting thatinnate and immovable disparity between donor and recipient, and thusfocusing on interventions, gene alterations, and/or concomitantexogenous immunosuppressive medications used as a method ofreducing/eliminating/negatively-altering the recipients' naturallyresulting immunologic response, shifting (if not reversing) the focus ofthe otherwise area of fundamental scientific dogma.

In certain other aspects, the present disclosure provides geneticallymodified, non-transgenic swine that are minimally altered. For example,in the present invention, certain distinct sequences appearing on thedonor swine SLA comprising native base pairs are removed and replacedwith a synthetic sequence comprising the same number of base pairs butreprogrammed based on the recipient's human capture sequence. Thisminimal alteration keeps other aspects of the native swine genome inplace and does not disturb, for example, introns and other codonsnaturally existing in the swine genome.

In certain other aspects, the present invention provides swine with suchand other modifications, created in a designated pathogen environment inaccordance with the processes and methods provided herein.

In certain other aspects the products derived from such swine forxenotransplantation are minimally manipulated, viable, live cell, andcapable of making an organic union with the transplant recipient,including, but not limited to, inducing vascularization and/or collagengeneration in the transplant recipient.

In certain other aspects products derived from such source animals arepreserved, including, but not limited to, through cryopreservation, in amanner that maintains viability and live cell characteristics of suchproducts.

In certain other aspects, such products are for homologous use, i.e.,the repair, reconstruction, replacement or supplementation of arecipient's organ, cell and/or tissue with a corresponding organ, celland/or tissue that performs the same basic function or functions as thedonor (e.g., swine skin is used as a transplant for human skin, swinekidney is used as a transplant for human kidney, swine liver is used asa transplant for human liver, swine nerve is used as a transplant forhuman nerve and so forth).

In certain other aspects, the present invention that the utilization ofsuch products in xenotransplantation be performed with or without theneed to use immunosuppressant drugs or therapies which inhibit orinterfere with normal immune function.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the disclosure, help illustrate various aspects of the presentinvention and, together with the description, further serve to describethe invention to enable a person skilled in the pertinent art to makeand use the aspects disclosed herein. In the drawings, like referencenumbers indicate identical or functionally similar elements.

FIG. 1 illustrates an image of human trophoblast and trophoblast cells.

FIG. 2 schematically illustrates a T Cell Receptor (TCR) binding MHCClass I and a peptide.

FIG. 3 schematically illustrates HLA Class I on the surface of a cell.

FIG. 4 schematically illustrates a Cytotoxic T Cell (CD8+)-Target CellInteraction.

FIG. 5 schematically illustrates a Cytotoxic T Cell (CD4+)-Target CellInteraction.

FIG. 6 schematically illustrates codominant expression of HLA genes andthe position of HLA genes on human chromosome 6.

FIG. 7 is a table listing numbers of serological antigens, proteins, andalleles for human MHC Class I and Class II isotypes.

FIG. 8 schematically illustrates HLA Class I and Class II on the surfaceof a cell.

FIG. 9 shows the structure of MHC Class I (A) and Class II proteins (B).The two globular domains furthest from the plasma membrane that form thepeptide binding region (PBR) are shaded in blue. The two Ig-likedomains, including the β2-microglobulin, are shaded in grey.

FIG. 10 shows the HLA genomic loci map.

FIG. 11 schematically illustrates Human MHC Class I and Class IIisotypes.

FIG. 12 shows the schematic molecular organization of the HLA Class Igenes. Exons are represented by the rectangles and introns by lines.

FIG. 13 shows the schematic molecular organization of the HLA Class IIgenes. Exons are represented by the rectangles and introns by lines.

FIG. 14 showing composite genetic alteration design for “humanization”of extracellular porcine cell expression

FIG. 15 shows comparative genomic organization of the human and swinemajor histocompatibility complex (MHC) Class I region. The humanleukocyte antigen (HLA) Class I map is adapted from Ref. [17] and theswine leukocyte antigen (SLA) Class I map is based only on one fullysequenced haplotype (Hp-1.1, H01) [4]. Note that not all the genes areshown here and the scale is approximate. The number and location ofexpressed SLA Class I genes may vary between haplotypes.

FIG. 16 shows comparative genomic organization of the human and swinemajor histocompatibility complex (MHC) Class II region. The humanleukocyte antigen (HLA) Class II map is adapted from Ref. [17] and theswine leukocyte antigen (SLA) Class II map is based only on one fullysequenced haplotype (H01) [4]. Note that not all the genes are shownhere and the scale is approximate. *The number and location of expressedHLA-DRB genes and pseudogenes may vary between haplotypes.

FIG. 17 shows a physical map of the SLA complex. Black boxes: locicontaining MHC-related sequences. White boxes: loci without MHC-relatedsequences. From the long arm to the short arm of the chromosome, theorder of the regions is Class II (II), Class III (III) and Class I (I).

FIG. 18 shows the schematic molecular organization of the SLA genes.Exons are represented by the gray ovals and introns by lines. Genelength is approximate to that found for the Hp-1.1 genome sequence.

FIG. 19 shows a side-by-side genomic analysis of the peptide sequences.

FIG. 20 shows the location and the length α1(exon 2) of SLA-DQA andβ1(exon 2) of SLA-DQB1.

FIG. 21 shows a spreadsheet detailing nucleotide sequences of exons andintrons of SLA-DQA and SLA-DQB1.

FIG. 22 shows SLA-DQ beta1 domain of Sus scrofa (wild boar).

FIG. 23 illustrates nomenclature of HLA alleles. Each HLA allele namehas a unique number corresponding to up to four sets of digits separatedby colons. The length of the allele designation is dependent on thesequence of the allele and that of its nearest relative. All allelesreceive at least a four digit name, which corresponds to the first twosets of digits, longer names are only assigned when necessary. Thedigits before the first colon describe the type, which often correspondsto the serological antigen carried by an allotype. The next set ofdigits are used to list the subtypes, numbers being assigned in theorder in which DNA sequences have been determined. Alleles whose numbersdiffer in the two sets of digits must differ in one or more nucleotidesubstitutions that change the amino acid sequence of the encodedprotein. Alleles that differ only by synonymous nucleotide substitutions(also called silent or non-coding substitutions) within the codingsequence are distinguished by the use of the third set of digits.Alleles that only differ by sequence polymorphisms in the introns, or inthe 5′ or 3′ untranslated regions that flank the exons and introns, aredistinguished by the use of the fourth set of digits.

FIG. 24 shows the length of exons and introns in HLA-DQA

FIG. 25A shows nucleotide sequence library between recipient specificHLA-DQA and HLA-DQA acquired from database, FIG. 25B shows NucleotideSequence Library identifying complete divergence between HLA vsSLA(DQ-A, Exon 2), FIG. 25C shows Human Capture Reference Sequence forDQ-A1 for Three Patients, FIG. 25D shows Human Capture ReferenceSequence for DQ-B1 for Three Patients, FIG. 25E shows Human CaptureReference Sequence for DR-A for Three Patients, FIG. 25F shows HumanCapture Reference Sequence for DQR-B1 for Three Patients.

FIG. 26A shows example of Human Capture Reference Sequence (DQ-A1) forThree Patients, FIG. 26B shows example of Human Capture ReferenceSequence (DQ-B1) for Three Patients, FIG. 26C shows example of HumanCapture Reference Sequence (DR-A) for Three Patients, FIG. 26D showsexample of Human Capture Reference Sequence (DR-B1) for Three Patients.

FIG. 27 shows the wild-type human beta-2 microglobulin protein andschematic molecular organization of the human B2M gene and swine B2Mgene.

FIG. 28 shows comparison of amino acid sequences of exon 2 of human B2Mvs exon 2 of swine B2M

FIG. 29 shows Phenotyping analysis of porcine alveolar macrophages(PAM). Cells were cultured in medium alone (control), or were activatedfor 72 hours with 100 ng/mL IFN-γ or loaded 30 μg/mL KLH for 24 hours.The cells were stained for SLA-DQ and marker is detected using antimouse APC-conjugated polyclonal IgG secondary antibody. Data ispresented as histograms of count (y axis) versus fluorescence intensityin log scale (x axis). Percentage of positive and negative cells forSLA-DQ for activated cells are shown on histograms.

FIGS. 30A-30B show SI values for BrdU ELISA. Proliferation response ofthree human CD4+ T cells (A) and PBMCs (B) to untreated and IFN-γactivated PAM cells (15K) after seven days incubation.

FIG. 31 shows a schematic depiction of a humanized porcine cellaccording to the present disclosure

FIGS. 32A-32B show SI values for BrdU ELISA. Proliferation response ofthree human CD4+ T cells (A) and PBMCs (B) to untreated and IFN-γactivated PAM cells (15K) after seven days incubation.

FIG. 33 shows schematic depiction of a humanized porcine cell accordingto the present disclosure.

FIG. 34 shows graph of proliferation of human plasma donors run on 3separate days with WT 128-11 and Gal T-KO B-174 PBMCs

FIG. 35 shows NK cytotoxicity of two donors (upper panel: KH; lowerpanel: MS) against 13 271 cells transfected with HLA-E/A2 (left column)and HLA-E/B7 (right column) compared to the lysis of untransfected 13271 cells. Results are depicted as percentage of specific lysis and wereobtained at four different E:T ratios. Data are representative of threeindependent experiments. Open triangles represent HLA-E-transfected 13271 cells, filled diamonds represent un-transfected 13 271 cells.(Forte, et al., 2005)

FIGS. 36A-36B show graphs of % cytotoxicity for each concentration(dilution) of plasma, and the results plotted in Prism. Based on thecytotoxicity curve, the required dilution for 50% kill (IC50) wasdetermined.

FIG. 37 illustrates a source animal facility and correspondingdesignated pathogen free facilities, animals, and herds in accordancewith the present invention.

FIG. 38 illustrates an extracorporeal liver filter and circuit inaccordance with the present invention.

FIG. 39 illustrates a combination skin product in accordance with thepresent invention.

FIG. 40A depicts POD-15. H&E, H&E, high power image depicts tissueviability with surface and follicular epithelial necrosis. FIG. 40Bdepicts POD-22 H&E, high power image demonstrating residual autograft(asterisks) with good overall viability. No surface epithelium and somesurface necrosis noted, along with extensive fibrosis with infiltrationinto the autograft (arrows).

FIG. 41 depicts longitudinal progression of porcine split-thickness skingraft used as a temporary wound closure in treatment of full-thicknesswound defects in a non-human primate recipient. Left: POD-0,xenotransplantation product at Wound Site 2. Right: POD-30, samexenotransplantation product at Wound Site 2.

FIG. 42 shows POD-30 histological images for: Top, Center: H&E, Lowpower image of wound site depicts complete epithelial coverage. Dottedline surrounds the residual xenotransplantation product.

FIG. 43A graphs the total serum IgM ELISA (μg/mL) for all four subjects(2001, 2002, 2101, 2102) during the course of the study. FIG. 43B graphsthe total serum IgG ELISA (μg/mL) for all four subjects (2001, 2002,2101, 2102) during the course of the study.

FIG. 44A graphs systemic concentrations of soluble CD40L as measured byLuminex 23-plex at POD-0, POD-7, POD-14, POD-21, and POD-30. FIG. 44Bgraphs systemic concentrations of TGF-alpha as measured by Luminex23-plex at POD-0, POD-7, POD-14, POD-21, and POD-30. FIG. 44C graphssystemic concentrations of IL-12/23 (p40) as measured by Luminex 23-plexat POD-0, POD-7, POD-14, POD-21, and POD-30.

FIG. 45 illustrates a method for preparing a skin product in accordancewith the present invention.

FIG. 46 shows a cryovial used to store a xenotransplantation product.

FIG. 47 shows a shipping process of a xenotransplantation product.

FIG. 48 shows a secondary closure or container system for storing axenotransplantation product at temperatures below ambient temperature,including, but not limited to, −150 degrees Celsius and othertemperatures.

FIG. 49A depicts porcine split-thickness skin grafts at wound sites 1,2, 3, and 4, respectively from left to right at POD-12. FIG. 49B depictsporcine split-thickness skin grafts at wound site 4 at POD-12 (left) andPOD-14 (right).

FIG. 50A graphs MTT reduction assays fresh vs. cryopreserved (7 years)in porcine tissue samples showing no statistical difference. FIG. 50Bgraphs MTT reduction assays heat deactivated vs. cryopreserved (7 years)in porcine tissue samples showing a statistically significant differentin quantity of formazan produced.

FIGS. 51A-51G show images of a xenotransplantation product of thepresent disclosure for treatment of severe and extensive partial andfull thickness burns in a human patient.

FIG. 52 shows a graph of proliferative response of human lymphocytesresponder peripheral blood mononuclear cells (PBMC) in the presence ofmitomycin C treated porcine stimulator cells.

FIG. 53 shows anti-xenogeneic IgM (A) and IgG (B) antibody binding datarelative to Median Fluorescence Intensities (MFI) for Xeno-001-00-1patient sample at multiple time points, Pre, Day 7, Day 16, and Day 30.The data is shown for the plasma samples tested at 1:2 dilutions.

DETAILED DESCRIPTION OF THE INVENTION

While aspects of the subject matter of the present disclosure may beembodied in a variety of forms, the following description is merelyintended to disclose some of these forms as specific examples of thesubject matter encompassed by the present disclosure. Accordingly, thesubject matter of this disclosure is not intended to be limited to theforms or aspects so described.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. Other features and advantages of theinvention will be apparent from the following detailed description andfigures, and from the claims.

“Best alignment” or “optimum alignment” means the alignment for whichthe identity percentage determined as described below is the highest.Comparisons of sequences between two nucleic acid sequences aretraditionally made by comparing these sequences after aligning themoptimally, the said comparison being made by segment or by “comparisonwindow” to identify and compare local regions for similar sequences. Forthe comparison, sequences may be optimally aligned manually, or by usingalignment software, e.g., Smith and Waterman local homology algorithm(1981), the Neddleman and Wunsch local homology algorithm (1970), thePearson and Lipman similarity search method (1988), and computersoftware using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTAand TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.). In some aspects, the optimumalignment is obtained using the BLAST program with the BLOSUM 62 matrixor software having similar functionality. The “identity percentage”between two sequences of nucleic acids or amino acids is determined bycomparing these two optimally aligned sequences, the sequence of nucleicacids or amino acids to be compared possibly including additions ordeletions from the reference sequence for optimal alignment betweenthese two sequences. The identity percentage is calculated bydetermining the number of positions for which the nucleotide or theamino acid residue is identical between the two sequences, by dividingthis number of identical positions by the total number of comparedpositions and multiplying the result obtained by 100 to obtain theidentity percentage between these two sequences.

“Conservative,” and its grammatical equivalents as used herein include aconservative amino acid substitution, including substitution of an aminoacid residue by another amino acid residue having a side chain R groupwith similar chemical properties (e.g., charge or hydrophobicity).Conservative amino acid substitutions may be achieved by modifying anucleotide sequence so as to introduce a nucleotide change that willencode the conservative substitution. In general, a conservative aminoacid substitution will not substantially change the functionalproperties of interest of a protein, for example, the ability of MHC Ito present a peptide of interest. Examples of groups of amino acids thathave side chains with similar chemical properties include aliphatic sidechains such as glycine, alanine, valine, leucine, and isoleucine;aliphatic-hydroxyl side chains such as serine and threonine;amide-containing side chains such as asparagine and glutamine; aromaticside chains such as phenylalanine, tyrosine, and tryptophan; basic sidechains such as lysine, arginine, and histidine; acidic side chains suchas aspartic acid and glutamic acid; and, sulfur-containing side chainssuch as cysteine and methionine. Conservative amino acids substitutiongroups include, for example, valine/leucine/isoleucine,phenylalanine/tyrosine, lysine/arginine, alanine/valine,glutamate/aspartate, and asparagine/glutamine. One skilled in the artwould understand that in addition to the nucleic acid residues encodinga human or humanized MHC I polypeptide and/or β2 microglobulin describedherein, due to the degeneracy of the genetic code, other nucleic acidsequences may encode the polypeptide(s) of the invention. Therefore, inaddition to a genetically modified non-human animal that comprises inits genome a nucleotide sequence encoding MHC I and/or β2 microglobulinpolypeptide(s) with conservative amino acid substitutions, a non-humananimal whose genome comprises a nucleotide sequence(s) that differs fromthat described herein due to the degeneracy of the genetic code is alsoprovided.

“Conserved” and its grammatical equivalents as used herein includenucleotides or amino acid residues of a polynucleotide sequence or aminoacid sequence, respectively, that are those that occur unaltered in thesame position of two or more related sequences being compared.Nucleotides or amino acids that are relatively conserved are those thatare conserved amongst more related sequences than nucleotides or aminoacids appearing elsewhere in the sequences. Herein, two or moresequences are said to be “completely conserved” if they are 100%identical to one another. In some embodiments, two or more sequences aresaid to be “highly conserved” if they are at least 70% identical, atleast 80% identical, at least 90% identical, or at least 95% identical,but less than 100% identical, to one another. In some embodiments, twoor more sequences are said to be “conserved” if they are at least 30%identical, at least 40% identical, at least 50% identical, at least 60%identical, at least 70% identical, at least 80% identical, at least 90%identical, or at least 95% identical, but less than 100% identical, toone another. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about50% identical, about 60% identical, about 70% identical, about 80%identical, about 90% identical, about 95% identical, about 98%identical, or about 99% identical to one another.

“Designated pathogen free,” and its grammatical equivalents as usedherein include reference to animals, animal herds, animal productsderived therefrom, and/or animal facilities that are free of one or morespecified pathogens. Preferably, such “designated pathogen free”animals, animal herds, animal products derived therefrom, and/or animalfacilities are maintained using well-defined routines of testing forsuch designated pathogens, utilizing proper standard operatingprocedures (SOPs) and practices of herd husbandry and veterinary care toassure the absence and/or destruction of such designated pathogens,including routines, testing, procedures, husbandry, and veterinary caredisclosed and described herein. It will be further understood that asused herein the term “free,” and like terms when used in connection with“pathogen free” are meant to indicate that the subject pathogens are notpresent, not alive, not active, or otherwise not detectable by standardor other testing methods for the subject pathogens.

“Alter,” “altering,” “altered” and grammatical equivalents as usedherein include any and/or all modifications to a gene including, but notlimited to, deleting, inserting, silencing, modifying, reprogramming,disrupting, mutating, rearranging, increasing expression, knocking-in,knocking out, and/or any or all other such modifications or anycombination thereof.

“Endogenous loci” and its grammatical equivalents as used herein includethe natural genetic loci found in the animal to be transformed into thedonor animal.

“Functional,” e.g., in reference to a functional polypeptide, and itsgrammatical equivalents as used herein include a polypeptide thatretains at least one biological activity normally associated with thenative protein. For example, in some embodiments of the invention, areplacement at an endogenous locus (e.g., replacement at an endogenousnon-human MHC I, MHC II, and/or β2 microglobulin locus) results in alocus that fails to express a functional endogenous polypeptide.Likewise, the term “functional” as used herein in reference tofunctional extracellular domain of a protein, can refer to anextracellular domain that retains its functionality, e.g., in the caseof MHC I, ability to bind an antigen, ability to bind a T cellco-receptor, etc. In some embodiments of the invention, a replacement atthe endogenous MHC locus results in a locus that fails to express anextracellular domain (e.g., a functional extracellular domain) of anendogenous MHC while expressing an extracellular domain (e.g., afunctional extracellular domain) of a human MHC.

“Genetic or molecular marker,” and their grammatical equivalents as usedherein include polymorphic locus, i.e. a polymorphic nucleotide (aso-called single nucleotide polymorphism or SNP) or a polymorphic DNAsequence at a specific locus. A marker refers to a measurable, geneticcharacteristic with a fixed position in the genome, which is normallyinherited in a Mendelian fashion, and which can be used for mapping of atrait of interest. Thus, a genetic marker may be a short DNA sequence,such as a sequence surrounding a single base-pair change, i.e. a singlenucleotide polymorphism or SNP, or a long DNA sequence, such asmicrosatellites or Simple Sequence Repeats (SSRs). The nature of themarker is dependent on the molecular analysis used and can be detectedat the DNA, RNA or protein level. Genetic mapping can be performed usingmolecular markers such as, but not limited to, RFLP (restrictionfragment length polymorphisms; Botstein et al. (1980), Am J Hum Genet.32:314-331; Tanksley et al. (1989), Bio/Technology 7:257-263), RAPD[random amplified polymorphic DNA; Williams et al. (1990), NAR18:6531-6535], AFLP [Amplified Fragment Length Polymorphism; Vos et al.(1995) NAR 23:4407-4414], SSRs or microsatellites [Tautz et al. (1989),NAR 17:6463-6471]. Appropriate primers or probes are dictated by themapping method used.

“Improving” and its grammatical equivalents as used herein include anyimprovement recognized by one of skill in the art. For example,improving transplantation can mean lessening hyperacute rejection, whichcan encompass a decrease, lessening, or diminishing of an undesirableeffect or symptom. In some aspects, a clinically relevant improvement isachieved.

“Locus” (loci plural) or “site” and their grammatical equivalents asused herein include a specific place or places on a chromosome where,for example, a gene, a genetic marker or a QTL is found.

“Minimally altered” and its grammatical equivalents as used hereininclude alteration of a donor animal genome including removing andreplacing certain distinct sequences of native base pairs appearing onthe donor animal's genome and replacing each such sequence with asynthetic sequence comprising the same number of base pairs, with no netchange to the number of base pairs in the donor animal's genome, whilenot disturbing other aspects of the donor animal's native genomeincluding, for example, introns and other codons naturally existing inthe donor animal genome. For example, in the case of a swine as donoranimal, a minimally altered swine can include specific alterationsremoving or deactivating certain SLA exons to regulate the donor swinecell's extracellular expression or non-expression of MHC Class II, Ia,and/or Ib; reprogramming certain native, naturally occurring swine cellSLA exons to regulate the swine cell's extracellular expression ornon-expression of MHC Class II; conserving or otherwise not removingswine introns existing in or in the vicinity of the otherwise engineeredsequences; increasing the expression of swine CTLA4 and PD-1; andremoving or deactivating alpha-1,3 galactosyltransferase, cytidinemonophosphate-N-acetylneuraminic acid hydroxylase, andβ1,4-N-acetylgalactosaminyltransferase.

“Minimally manipulated” and its grammatical equivalents as used hereininclude treatment of source animals, biological products derived fromthose source animals, and other biological products with minimalphysical alteration of the related cells, organs or tissues such thatsuch animals and products are substantially in their natural state.

“Ortholog,” “orthologous,” and their grammatical equivalents as usedherein include a polynucleotide from one species that corresponds to apolynucleotide in another species, which has the same function as thegene or protein or QTL, but is (usually) diverged in sequence from thetime point on when the species harboring the genes or quantitative traitloci diverged (i.e. the genes or quantitative trait loci evolved from acommon ancestor by speciation).

“Quantitative trait locus (QTL)” and its grammatical equivalents as usedherein include a stretch of DNA (such as a chromosome arm, a chromosomeregion, a nucleotide sequence, a gene, and the like) that is closelylinked to a gene that underlies the trait in question. “QTL mapping”involves the creation of a map of the genome using genetic or molecularmarkers, like AFLP, RAPD, RFLP, SNP, SSR, and the like, visiblepolymorphisms and allozymes, and determining the degree of associationof a specific region on the genome to the inheritance of the trait ofinterest. As the markers do not necessarily involve genes, QTL mappingresults involve the degree of association of a stretch of DNA with atrait rather than pointing directly at the gene responsible for thattrait. Different statistical methods are used to ascertain whether thedegree of association is significant or not. A molecular marker is saidto be “linked” to a gene or locus, if the marker and the gene or locushave a greater association in inheritance than would be expected fromindependent assortment, i.e. the marker and the locus co-segregate in asegregating population and are located on the same chromosome. “Linkage”refers to the genetic distance of the marker to the locus or gene (ortwo loci or two markers to each other). The closer the linkage, thesmaller the likelihood of a recombination event taking place, whichseparates the marker from the gene or locus. Genetic distance (mapdistance) is calculated from recombination frequencies and is expressedin centiMorgans (cM) [Kosambi (1944), Ann. Eugenet. 12:172-175].

“Capture sequence” or “reference sequence” and their grammaticalequivalents as used herein include a nucleic acid or amino acid sequencethat has been obtained, sequenced or otherwise become known from asample, animal (including humans), or population. For example, a capturesequence from a human patient is a “human patient capture sequence.” Acapture sequence from a particular human population is a “humanpopulation-specific human capture sequence.” And a capture sequence froma human allele group is an “allele-group-specific human capturesequence.”

“Humanized” and its grammatical equivalents as used herein includeembodiments wherein all or a portion of an endogenous non-human gene orallele is replaced by a corresponding portion of an orthologous humangene or allele. For example, in some embodiments, the term “humanized”refers to the complete replacement of the coding region (e.g., theexons) of the endogenous non-human MHC gene or allele or fragmentthereof with the corresponding capture sequence of the human MHC gene orallele or fragment thereof, while the endogenous non-coding region(s)(such as, but not limited to, the promoter, the 5′ and/or 3′untranslated region(s), enhancer elements, etc.) of the non-human animalis not replaced.

“Personalized” or “individualized,” and their grammatical equivalents asused herein, include a gene, allele, genome, proteome, cell, cellsurface, tissue, or organ from a non-human animal which is adapted tothe needs or special circumstances of an individual human recipient or aspecific human recipient subpopulation.

“Reprogram,” “reprogrammed,” including in reference to “immunogenomicreprogramming,” and their grammatical equivalents as used herein, referto the replacement or substitution of endogenous nucleotides in thedonor animal with orthologous nucleotides based on a separate referencesequence, wherein frameshift mutations are not introduced by suchreprogramming. In addition, reprogramming results in no net loss or netgain in the total number of nucleotides in the donor animal genome, orresults in a net loss or net gain in the total number of nucleotides inthe donor animal genome that is equal to no more than 1%, no more than2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%,no more than 7%, no more than 8%, no more than 9%, no more than 10%, nomore than 12%, no more than 15%, or no more than 20% of the number ofnucleotides in the separate reference sequence. In one example of“reprogramming,” an endogenous non-human nucleotide, codon, gene orfragment thereof is replaced with a corresponding synthetic nucleotide,codon, gene or fragment thereof based on a human capture sequence,through which the total number of base pairs in the donor animalsequence is equal to the total number of base pairs of the human capturesequence.

“Tolerogenic” and its grammatical equivalents as used herein includecharacteristics of an organ, cell, tissue, or other biological productthat are tolerated by the reduced response by the recipient's immunesystem upon transplantation.

“Transgenic” and its grammatical equivalents as used herein, includedonor animal genomes that have been modified to introduce non-nativegenes from a different species into the donor animal's genome at anon-orthologous, non-endogenous location such that the homologous,endogenous version of the gene (if any) is retained in whole or in part.“Transgene,” “transgenic,” and grammatical equivalents as used herein donot include reprogrammed genomes, knock-outs or other modifications asdescribed and claimed herein. By way of example, “transgenic” swineinclude those having or expressing hCD46 (“human membrane cofactorprotein,” or “MCP”), hCD55 (“human decay-accelerating factor,” “DAF”),human B2M (beta-2-microglobulin), and/or other human genes, achieved byinsertion of human gene sequences at a non-orthologous, non-endogenouslocation in the swine genome without the replacement of the endogenousversions of those genes.

Immunogenomic Reprogrammed Swine

As disclosed herein, tolerogenic non-human animal cells, tissues andorgans for several human Class I and/or Class II MHC molecules areprovided.

The human immune response system is a highly complex and efficientdefense system against invading organisms. T-cells are the primaryeffector cells involved in the cellular response. Just as antibodieshave been developed as therapeutics, (TCRs), the receptors on thesurface of the T-cells, which give them their specificity, have uniqueadvantages as a platform for developing therapeutics. While antibodiesare limited to recognition of pathogens in the blood and extracellularspaces or to protein targets on the cell surface, TCRs recognizeantigens displayed by MHC molecules on the surfaces of cells (includingantigens derived from intracellular proteins). Depending on the subtypeof T-cells that recognize displayed antigen and become activated, TCRsand T-cells harboring TCRs participate in controlling various immuneresponses. For instance, helper T-cells are involved in regulation ofthe humoral immune response through induction of differentiation of Bcells into antibody secreting cells. In addition, activated helperT-cells initiate cell-mediated immune responses by cytotoxic T-cells.Thus, TCRs specifically recognize targets that are not normally seen byantibodies and also trigger the T-cells that bear them to initiate widevariety of immune responses.

It will be understood, that T-cell recognizes an antigen presented onthe surfaces of cells by means of the TCRs expressed on their cellsurface. TCRs are disulfide-linked heterodimers, most consisting of αand β chain glycoproteins. T-cells use recombination mechanisms togenerate diversity in their receptor molecules similar to thosemechanisms for generating antibody diversity operating in B cells(Janeway and Travers, Immunobiology 1997). Similar to the immunoglobulingenes, TCR genes are composed of segments that rearrange duringdevelopment of T-cells. TCR polypeptides consist of variable, constant,transmembrane and cytoplasmic regions. While the transmembrane regionanchors the protein and the intracellular region participates insignaling when the receptor is occupied, the variable region isresponsible for specific recognition of an antigen and the constantregion supports the variable region-binding surface. The TCR α chaincontains variable regions encoded by variable (V) and joining (J)segments only, while the β chain contains additional diversity (D)segments.

Major histocompatibility complex Class I (MHCI) and Class II (MHCII)molecules display peptides on antigen-presenting cell surfaces forsubsequent T-cell recognition. See FIG. 2. Within the human population,allelic variation among the classical MHCI and II gene products is thebasis for differential peptide binding, thymic repertoire bias andallograft rejection. MHC molecules are cell-surface glycoproteins thatare central to the process of adaptive immunity, functioning to captureand display peptides on the surface of antigen-presenting cells (APCs).MHC Class I (MHCI) molecules are expressed on most cells, bindendogenously derived peptides with sizes ranging from eight to ten aminoacid residues and are recognized by CD8 cytotoxic T-lymphocytes (CTL).See FIG. 3 and FIG. 4. On the other hand, MHC Class II (MHCII) arepresent only on specialized APCs, bind exogenously derived peptides withsizes varying from 9 to 22 residues, and are recognized by CD4 helperT-cells. See FIG. 5. These differences indicate that MHCI and MHCIImolecules engage two distinct arms of the T-cell-mediated immuneresponse, the former targeting invasive pathogens such as viruses fordestruction by CD8 CTLs, and the latter inducing cytokine-basedinflammatory mediators to stimulate CD4 helper T-cell activitiesincluding B-cell activation, maturation and antibody production. In someaspects, the biological product of the present disclosure is notrecognized by CD8+ T cells, do not bind anti-HLA antibodies, and areresistant to NK-mediated lysis.

The human leukocyte antigen (HLA) system or complex is a gene complexencoding the major histocompatibility complex (MHC) proteins in humans.These cell-surface proteins are responsible for the regulation of theimmune system in humans. The HLA gene complex resides on a 3 Mbp stretchwithin chromosome 6p21. See FIG. 6. HLA genes are highly polymorphic,which means that they have many different alleles, allowing them tofine-tune the adaptive immune system. See FIG. 7. The proteins encodedby certain genes are also known as antigens, as a result of theirhistoric discovery as factors in organ transplants. Different classeshave different functions. See FIG. 8 and FIG. 9.

The HLA segment is divided into three regions (from centromere totelomere), Class II, Class III and Class I. See FIG. 10. Classical ClassI and Class II HLA genes are contained in the Class I and Class IIregions, respectively, whereas the Class III locus bears genes encodingproteins involved in the immune system but not structurally related toMHC molecules. The classical HLA Class I molecules are of three types,HLA-A, HLA-B and HLA-C. Only the α chains of these mature HLA Class Imolecules are encoded within the Class I HLA locus by the respectiveHLA-A, HLA-B and HLA-C genes. See FIG. 11. In contrast, the beta-2microglobulin β2m chain encoded by the β2m gene is located on chromosome15. The classical HLA Class II molecules are also of three types(HLA-DP, HLA-DQ and HLA-DR), with both the α and β chains of eachencoded by a pair of adjacent loci. In addition to these classical HLAClass I and HLA Class II genes, the human MHC locus includes a longarray of HLA pseudogenes as well as genes encoding non-classical MHCIand MHCII molecules. HLA-pseudogenes are an indication that geneduplication is the main driving force for HLA evolution, whereasnon-classical MHCI and MHCII molecules often serve a restricted functionwithin the immune system quite distinct from that of antigenpresentation to αβ TCRs.

Aside from the genes encoding the antigen-presenting proteins, there area large number of other genes, many involved in immune function, locatedon the HLA complex. Diversity of HLAs in the human population is oneaspect of disease defense, and, as a result, the chance of two unrelatedindividuals with identical HLA molecules on all loci is extremely low.HLA genes have historically been identified as a result of the abilityto successfully transplant organs between HLA-similar individuals.

Class I MHC molecules are expressed on all nucleated cells, includingtumor cells. They are expressed specifically on T and B lymphocytes,macrophages, dendritic cells and neutrophils, among other cells, andfunction to display peptide fragments (typically 8-10 amino acids inlength) on the surface to CD8+ cytotoxic T lymphocytes (CTLs). CTLs arespecialized to kill any cell that bears an MHC I-bound peptiderecognized by its own membrane-bound TCR. When a cell displays peptidesderived from cellular proteins not normally present (e.g., of viral,tumor, or other non-self origin), such peptides are recognized by CTLs,which become activated and kill the cell displaying the peptide.

As shown in FIG. 12, MHC Class I protein comprises an extracellulardomain (which comprises three domains: α₁, α₂ and α₃), a transmembranedomain, and a cytoplasmic tail. The α₁ and α₂ domains form thepeptide-binding cleft, while the α₃ interacts with β2-microglobulin.Class I molecules consist of two chains: a polymorphic α-chain(sometimes referred to as heavy chain) and a smaller chain calledβ2-microglobulin (also known as light chain), which is generally notpolymorphic. These two chains form a non-covalent heterodimer on thecell surface. The α-chain contains three domains (α1, α2 and α3). Asillustrated in FIG. 12, Exon 1 of the α-chain gene encodes the leadersequence, exons 2 and 3 encode the α1 and α2 domains, exon 4 encodes theα3 domain, exon 5 encodes the transmembrane domain, and exons 6 and 7encode the cytoplasmic tail. The α-chain forms a peptide-binding cleftinvolving the α1 and α2 domains (which resemble Ig-like domains)followed by the α3 domain, which is similar to β2-microglobulin.

β2 microglobulin is a non-glycosylated 12 kDa protein; one of itsfunctions is to stabilize the MHC Class I α-chain. Unlike the α-chain,the β2 microglobulin does not span the membrane. The human β2microglobulin locus is on chromosome 15 and consists of 4 exons and 3introns. Circulating forms of β2 microglobulin are present in serum,urine, and other body fluids; non-covalently MHC I-associated β2microglobulin can be exchanged with circulating (32 microglobulin underphysiological conditions.

As shown in FIG. 13, MHC Class II protein comprises an extracellulardomain (which comprises three domains: α₁, α₂, β1, and β1), atransmembrane domain, and a cytoplasmic tail. The α₁ and β1 domains formthe peptide-binding cleft, while the α₁ and β1 interacts with thetransmembrane domain.

In addition to the aforementioned antigens, the Class I antigens includeother antigens, termed non-classical Class I antigens, in particular theantigens HLA-E, HLA-F and HLA-G; this latter, in particular, isexpressed by the extravillous trophoblasts of the normal human placentain addition to HLA-C.

Cell Phenotype

Referring generally to FIG. 1, Dr. Peter Medawar profoundly said “thesuccess of human pregnancy, where the fetus resides comfortably withinthe maternal uterus for 9 months, defies the precepts of immunology.”Paraphrasing, he observed that the most common, successful transplant onearth is pregnancy.

The trophoblast expression of cell surface markers is wellcharacterized, and by replicating such phenotype in the porcine cellwhere appropriate and necessary to retain, critical and desired cellularfunction can be obtained. According to literature, extravilloustrophoblast cells express HLA Class Ia molecule (HLA-C) and all of HLAClass Ib molecules. Compared to HLA-E and HLA-G, both of which arehighly expressed on extravillous trophoblast cells, HLA-C and HLA-F areweakly expressed. See, e.g., Djurisic et al., “HLA Class Ib Moleculesand Immune Cells in Pregnancy and Preeclampsia,” Frontiers inImmunology, Vol 5, Art. 652 (2014). In addition to MHC molecules, PD-L1is upregulated in trophoblastic cells in normal pregnancy, particularlyin syncytiotrophoblast cells. HLA Class II molecules are not present ontrophoblasts, which may facilitate survival and detection of the embryoin the presence of maternal lymphocytes. See, e.g., Veras et al., “PD-L1Expression in Human Placentas and gestational Trophoblastic Diseases,”Int. J. Gynecol. Pathol. 36(2): 146-153 (2017).

The present invention provides a method of creating a tolerogenicxenotransplantation swine cell that mimics the extracellularconfiguration of a human trophoblast. This method includes, but is notlimited to, removing or deactivating certain SLA exons to regulate theswine cell's extracellular expression or non-expression of MHC Class II,Ia, and/or Ib; reprogramming certain native, naturally occurring swinecell SLA exons to regulate the swine cell's extracellular expression ornon-expression of MHC Class II; conserving or otherwise not removingswine introns existing in or in the vicinity of the otherwise engineeredsequences; increasing the expression of swine CTLA4 and PD-1; andremoving or deactivating alpha-1,3 galactosyltransferase, cytidinemonophosphate-N-acetylneuraminic acid hydroxylase, andβ1,4-N-acetylgalactosaminyltransferase. Such removal, reprogramming, andmodification to cause such increase of expression, and other engineeredaspects of a swine genome, to create a tolerogenic xenotransplantationswine cell that mimics the extracellular configuration of a humantrophoblast, is described as follows.

The former and current attempts to this unmet clinical need hasprecisely followed the classic medical dogma of “one-size fits all”. Werefer to this as the “downstream” approach—which must contend withaddressing all of the natural immune processes in sequence. Instead ofadopting this limited view, the present invention takes a“patient-specific” solution to dramatically improve clinical outcomemeasures. The latter, our approach, we term the “upstream” approach—onewhich represents the culmination of unfilled scientific effort into acoordinated translational effort. The central theorem of our approach iscountervailing to the existing and previous dogmatic approaches. The“downstream” approach accepts the innate and immovable disparity betweendonor and recipient, and focuses on interventions, gene alterations,and/or concomitant exogenous immunosuppressive medications used as amethod of reducing/eliminating/negatively-altering the recipients'naturally resulting immunologic response. In contrast, we intentionallychoose to reverse the focus of the otherwise area of fundamentalscientific dogma. Rather than accept the immunological incompatibilitiesbetween the donor and recipient, specifically (but not limited to) thosemismatches of the Major Histocompatibility Complex(es), we alter thesecatalytic antigens at the source, thereby eliminating all of theprecipitating mechanisms that are the causative effectors of cell,tissue, and organ rejection between donor and recipient. This approachapplies beyond the field of xenotransplantation including, but notlimited to, the fields of genetics, obstetrics, infectious disease,oncology, agriculture, animal husbandry, food industry and other areas.

The present disclosure embodies the above modification in creating anon-transgenic genetically reprogrammed swine for xenotransplantation,wherein the MHC surface characterization of the swine mimic that of therecipient's trophoblast, wherein the immune response from thexenotransplantation is significantly reduced. The human extravilloustrophoblast cells express HLA-C, HLA-E, HLA-F, and HLA-G, but not HLA-A,HLA-B, HLA-DQ and HLA-DR. As such, the current embodiment combines theunique MHC surface characterization of human trophoblast withsite-directed mutagenic substitutions to minimize or remove the immuneresponse associated with xenotransplantation while minimizing off targeteffects on the native donor swine's SLA/MHC gene.

The human immune response system is a highly complex and efficientdefense system against invading organisms. T-cells are the primaryeffector cells involved in the cellular response. Just as antibodieshave been developed as therapeutics, (TCRs), the receptors on thesurface of the T-cells, which give them their specificity, have uniqueadvantages as a platform for developing therapeutics. While antibodiesare limited to recognition of pathogens in the blood and extracellularspaces or to protein targets on the cell surface, TCRs recognizeantigens displayed by MHC molecules on the surfaces of cells (includingantigens derived from intracellular proteins). Depending on the subtypeof T-cells that recognize displayed antigen and become activated, TCRsand T-cells harboring TCRs participate in controlling various immuneresponses. For instance, helper T-cells are involved in regulation ofthe humoral immune response through induction of differentiation of Bcells into antibody secreting cells. In addition, activated helperT-cells initiate cell-mediated immune responses by cytotoxic T-cells.Thus, TCRs specifically recognize targets that are not normally seen byantibodies and also trigger the T-cells that bear them to initiate widevariety of immune responses.

As shown in FIG. 2, a T-cell recognizes an antigen presented on thesurfaces of cells by means of the TCRs expressed on their cell surface.TCRs are disulfide-linked heterodimers, most consisting of α and β chainglycoproteins. T-cells use recombination mechanisms to generatediversity in their receptor molecules similar to those mechanisms forgenerating antibody diversity operating in B cells (Janeway and Travers,Immunobiology 1997). Similar to the immunoglobulin genes, TCR genes arecomposed of segments that rearrange during development of T-cells. TCRpolypeptides consist of variable, constant, transmembrane andcytoplasmic regions. While the transmembrane region anchors the proteinand the intracellular region participates in signaling when the receptoris occupied, the variable region is responsible for specific recognitionof an antigen and the constant region supports the variableregion-binding surface. The TCR α chain contains variable regionsencoded by variable (V) and joining (J) segments only, while the β chaincontains additional diversity (D) segments.

A TCR recognizes a peptide antigen presented on the surfaces of antigenpresenting cells in the context of self-Major Histocompatibility Complex(MHC) molecules. Two different types of MHC molecules recognized by TCRsare involved in antigen presentation, the Class I MHC and class II MHCmolecules. Mature T-cell subsets are defined by the co-receptormolecules they express. These co-receptors act in conjunction with TCRsin the recognition of the MHC-antigen complex and activation of theT-cell. Mature helper T-cells recognize antigen in the context of MHCClass II molecules and are distinguished by having the co-receptor CD4.Cytotoxic T-cells recognize antigen in the context of MHC Class Ideterminants and are distinguished by having the CD8 co-receptor.

In the human, MHC molecules are referred to as HLA, an acronym for humanleukocyte antigens, and are encoded by the chromosome 6p21.3-located HLAregion.8,9 The HLA segment is divided into three regions (fromcentromere to telomere), Class II, Class III and Class I. See FIG. 10.Classical Class I and Class II HLA genes are contained in the Class Iand Class II regions, respectively, whereas the Class III locus bearsgenes encoding proteins involved in the immune system but notstructurally related to MHC molecules. The classical HLA Class Imolecules are of three types, HLA-A, HLA-B and HLA-C. Only the α chainsof these mature HLA Class I molecules are encoded within the Class I HLAlocus by the respective HLA-A, HLA-B and HLA-C genes. See FIG. 11. Incontrast, the beta-2 microglobulin β2m chain encoded by the β2m gene islocated on chromosome 15. The classical HLA Class II molecules are alsoof three types (HLA-DP, HLA-DQ and HLA-DR), with both the α and β chainsof each encoded by a pair of adjacent loci. In addition to theseclassical HLA Class I and HLA Class II genes, the human MHC locusincludes a long array of HLA pseudogenes as well as genes encodingnon-classical MHCI and MHCII molecules. HLA-pseudogenes are anindication that gene duplication is the main driving force for HLAevolution, whereas non-classical MHCI and MHCII molecules often serve arestricted function within the immune system quite distinct from that ofantigen presentation to αβ TCRs.

Human leukocyte antigen (HLA) genes show incredible sequence diversityin the human population. For example, there are >4,000 known alleles forthe HLA-B gene alone. The genetic diversity in HLA genes in whichdifferent alleles have different efficiencies for presenting differentantigens is believed to be a result of evolution conferring betterpopulation-level resistance against the wide range of differentpathogens to which humans are exposed. This genetic diversity alsopresents problems during xenotransplantation where the recipient'simmune response is the most important factor dictating the outcome ofengraftment and survival after transplantation.

In humans, the classical Class I genes, termed HLA-A, HLA-B and HLA-C,consist of two chains: a polymorphic α-chain (sometimes referred to asheavy chain) and a smaller chain called β2-microglobulin (also known aslight chain), which is generally not polymorphic. These two chains forma non-covalent heterodimer on the cell surface. As shown in FIG. 12, theα-chain contains three domains (α1, α2 and α3). Exon 1 of the α-chaingene encodes the leader sequence, exons 2 and 3 encode the α1 and α2domains, exon 4 encodes the α3 domain, exon 5 encodes the transmembranedomain, and exons 6 and 7 encode the cytoplasmic tail. The α-chain formsa peptide-binding cleft involving the α1 and α2 domains (which resembleIg-like domains) followed by the α3 domain, which is similar toβ2-microglobulin.

β2 microglobulin is a non-glycosylated 12 kDa protein; one of itsfunctions is to stabilize the MHC Class I α-chain. Unlike the α-chain,the β2 microglobulin does not span the membrane. The human β2microglobulin locus is on chromosome 15 and consists of 4 exons and 3introns. β2-microglobulin-bound protein complexes undertake key roles invarious immune system pathways, including the neonatal Fc receptor(FcRn), cluster of differentiation 1 (CD1) protein, non-classical majorhistocompatibility complex (MHC), and well-known MHC Class I molecules.

Class I MHC molecules are expressed on all nucleated cells, includingtumor cells. They are expressed specifically on T and B lymphocytes,macrophages, dendritic cells and neutrophils, among other cells, andfunction to display peptide fragments (typically 8-10 amino acids inlength) on the surface to CD8+ cytotoxic T lymphocytes (CTLs). CTLs arespecialized to kill any cell that bears an MHC I-bound peptiderecognized by its own membrane-bound TCR. When a cell displays peptidesderived from cellular proteins not normally present (e.g., of viral,tumor, or other non-self origin), such peptides are recognized by CTLs,which become activated and kill the cell displaying the peptide.

MHC loci exhibit the highest polymorphism in the genome. All Class I andII MHC genes can present peptide fragments, but each gene expresses aprotein with different binding characteristics, reflecting polymorphismsand allelic variants. Any given individual has a unique range of peptidefragments that can be presented on the cell surface to B and T cells inthe course of an immune response.

In addition to the aforementioned antigens, the Class I antigens includeother antigens, termed non-classical Class I antigens, in particular theantigens HLA-E, HLA-F and HLA-G; this latter, in particular, isexpressed by the extravillous trophoblasts of the normal human placentain addition to HLA-C.

MHC Class II protein comprises an extracellular domain (which comprisesthree domains: α1, α2, β1, and β1), a transmembrane domain, and acytoplasmic tail as shown in FIG. 13. The α2 and β2 domains form thepeptide-binding cleft, while the α1 and β1 interacts with thetransmembrane domain.

With respect to the MHC-I proteins, the current disclosure eitherinactivate, or where necessary to retain the function of the “find andreplace” orthologous SLA proteins with HLA analogs that would result inminimal immune recognition. In some aspects, silencing the genes whichencode and are responsible for the expression of SLA-1 removes thehighly-problematic and polymorphic HLA-A analog. Similarly, inactivationor complete removal of genes associated with SLA-2 would reduce theburden imposed by mismatched HLA-B proteins. This would, at the cellsurface interface, appear to the human recipient's T cells as a HLA-Aand HLA-B negative cell. With respect to the last of the classical MHCClass I proteins, HLA-C, site-directed mutagenesis of genes that encodefor SLA-3 using a reference HLA-C sequence would mimic anallo-transplant with such a disparity. Given the “less-polymorphic”nature of HLA-C, as compared to HLA-A and HLA-B, this would be furtherimproved by the replacement of SLA-3 with a reference replacementsequence based on the subclass of HLA-C that is naturally prevalent innature, and also invoking mechanisms that would allow for the minimalbut requisite level of expression that would afford functionality andnon-interruption of the numerous known and also those unknown MHC-Idependent processes.

With respect to the MHC-I proteins, the current disclosure eitherinactivate, or where necessary to retain the function of the “find andreplace” orthologous SLA proteins with HLA analogs that would result inminimal immune recognition. In some aspects, silencing the genes whichencode and are responsible for the expression of SLA-1 removes thehighly-problematic and polymorphic HLA-A analog. Similarly, inactivationor complete removal of genes associated with SLA-2 would reduce theburden imposed by mismatched HLA-B proteins. This would, at the cellsurface interface, appear to the human recipient's T cells as a HLA-Aand HLA-B negative cell. With respect to the last of the classical MHCClass I proteins, HLA-C, site-directed mutagenesis of genes that encodefor SLA-3 using a reference HLA-C sequence would mimic anallo-transplant with such a disparity. Given the “less-polymorphic”nature of HLA-C, as compared to HLA-A and HLA-B, this would be furtherimproved by the replacement of SLA-3 with a reference replacementsequence based on the subclass of HLA-C that is naturally prevalent innature, and also invoking mechanisms that would allow for the minimalbut requisite level of expression that would afford functionality andnon-interruption of the numerous known and also those unknown MHC-Idependent processes.

Furthermore, the expression of non-classical MHC proteins—those includedin the I-b category, which include HLA-E, F, and G are vitally importantto both the survival of the fetus and synergistic existence of thetrophoblast(s). Fortunately, these are significantly less polymorphicthan the “classical” MHC-Ia variety. Without expression of these,heightened upregulation of cell lysis is a direct result of NK cellrecognition and activation is observed. In an identical manner asdescribed to the MHC-Ia components, the orthologous SLA proteins withHLA analogs are either inactivated, or where necessary, to “find andreplace(d)” FIG. 14 shows specific alterations that are included in thepresent disclosure.

HLA-G can be a potent immuno-inhibitory and tolerogenic molecule. HLA-Gexpression in a human fetus can enable the human fetus to elude thematernal immune response. Neither stimulatory functions nor responses toallogeneic HLA-G have been reported to date. HLA-G can be anon-classical HLA Class I molecule. It can differ from classical MHCClass I molecules by its genetic diversity, expression, structure, andfunction. HLA-G can be characterized by a low allelic polymorphism.Expression of HLA-G can be restricted to trophoblast cells, adult thymicmedulla, and stem cells. The sequence of the HLA-G gene (HLA-6.0 gene)has been described by GERAGHTY et al., (Proc. Natl. Acad. Sci. USA,1987, 84, 9145-9149): it comprises 4,396 base pairs and exhibits anintron/exon organization which is homologous to that of the HLA-A, HLA-Band HLA-C genes. More precisely, this gene comprises 8 exons and anuntranslated, 3′UT, end, with the following respective correspondence:exon 1: signal sequence, exon 2: α1 domain, exon 3: α2 domain, exon 4:α3 domain, exon 5: transmembrane region, exon 6: cytoplasmic domain I,exon 7: cytoplasmic domain II, exon 8: cytoplasmic domain III and 3′untranslated region (GERAGHTY et al., mentioned above, ELLIS et al., J.Immunol., 1990, 144, 731-735). However, the HLA-G gene differs from theother Class I genes in that the in-frame translation termination codonis located at the second codon of exon 6; as a result, the cytoplasmicregion of the protein encoded by this gene HLA-6.0 is considerablyshorter than that of the cytoplasmic regions of the HLA-A, HLA-B andHLA-C proteins.

Natural killer (NK) cell-mediated immunity, comprising cytotoxicity andcytokine secretion, plays a major role in biological resistance to anumber of autologous and allogeneic cells. The common mechanism oftarget cell recognition appears to be the lack or modification of selfMHC Class I-peptide complexes on the cell surface, which can lead to theelimination of virally infected cells, tumor cells and majorhistocompatibility MHC-incompatible grafted cells. KIR's, members of theIg superfamily which are expressed on NK cells, have recently beendiscovered and cloned. KIR's are specific for polymorphic MHC Class Imolecules and generate a negative signal upon ligand binding which leadsto target cell protection from NK cell-mediated cytotoxicity in mostsystems. In order to prevent NK cell autoimmunity, i.e., the lysis ofnormal autologous cells, it is believed that every given NK cell of anindividual expresses at least on KIR recognizing at least one of theautologous HLA-A, B, C, or G alleles.

According to the present disclosure, in the context of swine-to-humanxenotransplantation, each human recipient will have a majorhistocompatibility complex (MHC) (Class I, Class II and/or Class III)that is unique to that individual and will not match the MHC of thedonor swine. Accordingly, when a donor swine graft is introduced to therecipient, the swine MHC molecules themselves act as antigens, provokingan immune response from the recipient, leading to transplant rejection.

According to this aspect of the present disclosure (i.e., reprogrammingthe SLA/MHC to express specifically selected human MHC alleles), whenapplied to swine cells, tissues, and organs for purposes ofxenotransplantation will decrease rejection as compared to cells,tissues, and organs derived from a wild-type swine or otherwisegenetically modified swine that lacks this reprogramming, e.g.,transgenic swine or swine with non-specific or different geneticmodifications.

With the previous modifications incorporated, insertion or activation ofadditional extracellular ligands that would create a protective,localized immune response as seen with the maternal-fetal symbiosis,would be an additional step to minimize deleterious cellular-mediatedimmunological functions that may remain as a result of minor-antigendisparities. Therefore, porcine ligands for SLA-MIC2 is orthologouslyreprogrammed with human counterparts, MICA. Human MajorHistocompatibility Complex Class I Chain-Related gene A (MICA) is a cellsurface glycoprotein expressed on endothelial cells, dendritic cells,fibroblasts, epithelial cells, and many tumours. It is located on theshort arm of human chromosome 6 and consists of 7 exons, 5 of whichencodes the transmembrane region of the MICA molecule. MICA protein atnormal states has a low level of expression in epithelial tissues but isupregulated in response to various stimuli of cellular stress. MICA isclassified as a non-classical MHC Class I gene, and functions as aligand recognized by the activating receptor NKG2D that is expressed onthe surface of NK cells and CD8+ T cells(atlasgeneticsoncology.org/Genes/MICAID41364ch6p21.html).

In addition, porcine ligands for PD-L1, CTLA-4, and others areoverexpressed and/or otherwise orthologously reprogrammed with humancounterparts. PD-L1 is a transmembrane protein that has major role insuppressing the adaptive immune system in pregnancy, allografts, andautoimmune diseases. It is encoded by the CD274 gene in human and islocated in chromosome 9. PD-L1 binds to PD-1, a receptor found onactivated T cells, B cells, and myeloid cells, to modulate activation orinhibition. Particularly, the binding of PD-L1 to receptor PD-1 on Tcells inhibits activation of IL-2 production and T cell proliferation.CTLA4 is a protein receptor that also functions as an immune checkpointthat downregulates immune responses. It is encoded by the CTLA4 gene andis located in chromosome 2 in human. It is constitutively expressed onregulatory T cells but are upregulated in activated T cells. Geneexpression for CTLA-4 and PD-L1 is increased, for example, based onreprogramming promoters thereof. There is a relationship betweengenotype and CTLA-4 or PD-L1 expression. For example, individualscarrying thymine at position −318 of the CTLA4 promoter (T(−318)) andhomozygous for adenine at position 49 in exon 1 showed significantlyincreased expression both of cell-surface CTLA-4 after cellularstimulation and of CTLA-4 mRNA in non-stimulated cells in Ligers A, etal. CTLA-4 gene expression is influenced by promoter and exon 1polymorphisms, Genes Immun. 2001 May; 2(3):145-52, which is incorporatedherein by reference in its entirety for all purposes. A similarupregulation can be achieved to overexpress PD-L1 using a PD-L1 promoterreprogramming.

Further, anti-coagulant porcine ligands for Endothelial protein Creceptor (EPCR), Thrombomodulin (TBM), Tissue Factor Pathway Inhibitor(TFPI), and others are orthologously reprogrammed with humancounterparts, as shown in FIG. 14. Endothelial protein C receptor isendothelial cell-specific transmembrane glycoprotein encoded by PROCRgene that is located in chromosome 20 in human. It enhances activationof Protein C, an anti-coagulant serine protease, and has crucial role inactivated protein C mediated cytoprotive signaling. Thrombomodulin is anintegral membrane glycoprotein present on surface of endothelial cells.It is encoded by THBD gene that is located in chromosome 20 in human. Inaddition to functioning as cofactor in the thrombin-induced activationof protein C in the anticoagulant pathway, it also functions inregulating C3b inactivation. Tissue Factor Pathway Inhibitor (TFPI) is aglycoprotein that functions as natural anticoagulant by inhibitingFactor Xa. It encoded by TFPI gene located in chromosome 2 in human andthe protein structure consists of three tandemly linked Kunitz domains.In human, two major isoforms of TFPi exists, TFPIα and TFPIβ. TFPIαconsists of three inhibitory domains (K1, K2, and K3) and a positivelycharged C terminus while TFPIβ consists of two inhibitory domains (K1and K2) and C terminus. While K1 and K2 domains are known to bind andinhibit Factor VII and Factor Xa, respectively, the inhibitory functionof K3 is unknown. In certain aspects, the present disclosure centralizes(predicates) the creation of hypoimmunogenic and/or tolerogenic cells,tissues, and organs that does not necessitate the transplant recipients'prevalent and deleterious use of exogenous immunosuppressive drugs (orprolonged immunosuppressive regimens) following the transplant procedureto prolong the life-saving organ.

The table provided in FIG. 14 shows conceptual design that exhibitsummation of various edits to create tolerogenic xenotransplantationswine cell that mimics the extracellular configuration of a humantrophoblast. As exhibited in the FIG. 14, SLA-1, a swine geneorthologous to HLA-A, is silenced to mimic trophoblast, as HLA-A is notexpressed on trophoblast. As further exhibited in the FIG. 14, SLA-8, aswine gene orthologous to HLA-G, is humanized through replacement with“human-capture” reference sequence, as HLA-G is expressed in trophoblastand has crucial role in maternal fetal tolerance, given its interactionwith NK cells.

It is therefore understood that multiple source animals, with an arrayof biological properties including, but not limited to, genomemodification and/or other genetically engineered properties, can beutilized to reduce immunogenicity and/or immunological rejection (e.g.,acute, hyperacute, and chronic rejections) in humans resulting fromxenotransplantation. In certain aspects, the present disclosure can beused to reduce or avoid thrombotic microangiopathy by transplanting thebiological product of the present disclosure into a human patient. Incertain aspects, the present disclosure can be used to reduce or avoidglomerulopathy by transplanting the biological product of the presentdisclosure into a human patient. It will be further understood that thelisting of source animals set forth herein is not limiting, and thepresent invention encompasses any other type of source animal with oneor more modifications (genetic or otherwise) that serve(s) to reduceimmunogenicity and/or immunological rejection, singularly or incombination.

Bioinformatic Sequence Analysis Comparing Identities of Conserved andNon-Conserved Nucleotides Between Human Versus Swine Genomes at VariousImmunologically Critical Loci

To reprogram the MHC disparities between the Swine Leukocyte Antigen(SLA) and the Human Leukocyte Antigen (HLA), the present disclosureincludes using highly conserved MHC-loci between these two species,e.g., numerous genes that correspond in function. The MHC Class Ia,HLA-A, HLA-B, and HLA-C have an analogous partner in the swine (the SLA1, 2 and 3 respectively). In MHC Class II there are also numerousmatches to be utilized during immunogenomic reprogramming according tothe present disclosure.

As illustrated in FIG. 15, MHC genes are categorized into three classes;Class I, Class II, and Class III, all of which are encoded on humanchromosome 6. The MHC genes are among the most polymorphic genes of theswine and human genomes, MHC polymorphisms are presumed to be importantin providing evolutionary advantage; changes in sequence can result indifferences in peptide binding that allow for better presentation ofpathogens to cytotoxic T cells.

The known human HLA/MHC or an individual recipient's sequenced HLA/MHCsequence(s) may be utilized as a template to reprogram with precisesubstitution the swine leukocyte antigen (SLA)/MHC sequence to match,e.g., to have 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence homology toa known human HLA/MHC sequence or the human recipient's HLA/MHCsequence. Upon identifying a known human recipient HLA/MHC sequence tobe used or performing genetic sequencing of a human recipient to obtainHLA/MHC sequences, 3 reprogramming may be performed to SLA/MHC sequencesin cells of the swine based on desired HLA/MHC sequences. For example,several targeting guide RNA (gRNA) sequences are administered to theswine of the present disclosure to reprogram SLA/MHC sequences in cellsof the swine with the template HLA/MHC sequences of the human recipient.

The term “MHC I complex” or the like, as used herein, includes thecomplex between the MHC I α chain polypeptide and the β2-microglobulinpolypeptide. The term “MHC I polypeptide” or the like, as used herein,includes the MHC I α chain polypeptide alone. Typically, the terms“human MHC” and “HLA” can be used interchangeably.

For purposes of modifying donor SLA/MHC to match recipient HLA/MHC,comparative genomic organization of the human and swinehistocompatibility complex has been mapped as illustrated in FIG. 16 andFIG. 17. For example, such SLA to HLA mapping can be found in: Lunney,J., “Molecular genetics of the swine major histocompatibility complex,the SLA complex,” Developmental and Comparative Immunology 33: 362-374(2009) (“Lunney”), the entire disclosure of which is incorporated hereinby reference. Further, by comparing the loci of HLA and schematicmolecular organization of various HLA genes, as illustrated in FIG. 12and FIG. 13, with the loci of SLA and schematic molecular organizationof various SLA genes, as show in FIG. 17 and FIG. 18, it is readilydiscernible that the placement and number of exons in extracellular andtransmembrane domain is common between HLA MHC and SLA MHC. Accordingly,a person of ordinary skill in the art effectively and efficientlygenetically reprogram swine cells in view of the present disclosure andusing the mapping of Lunney et al. as a reference tool.

The donor swine's SLA/MHC gene is used as a reference template increating the replacement template. In implementing the presentdisclosure, the swine's SLA/MHC gene may be obtained through onlinearchives or database such as Ensembl(http://vega.archive.ensembl.org/index.html). As illustrated in FIG. 19,FIG. 20, FIG. 21, and FIG. 22, the exact location of the SLA-DQA andSLA-DQB1 gene, the length of the respective gene (exon and intron), andthe exact nucleotide sequences of SLA-DQA and SLA-DQB1 are mapped. In analternative aspect of the present disclosure, the donor swine's SLA/MHCgene may be sequenced. In an alternative aspect of the presentdisclosure, the swine's whole genome may be sequenced. In one aspect,the sequenced SLA/MHC gene of the donor swine that can be used as areference template include but are not limited to SLA-3, SLA-6, SLA-7,SLA-8, SLA-DQa, SLA-DQb, and beta-2 microglobulin. In another aspect,the sequenced SLA/MHC gene of the donor swine that can be used as a basetemplate include but are not limited exon regions of SLA-3, SLA-6,SLA-7, SLA-8, SLA-DQa, SLA-DQb, and beta-2 microglobulin. In someaspects, other SLAs are unaltered and intron regions of the reprogrammedSLA regions are unaltered, thereby producing a minimally alteredreprogrammed swine genome that provides cells, tissues and organs thatare tolerogenic when transplanted into a human.

In accordance with one aspect the present invention, a donor swine isprovided with a genome that is biologically engineered to express aspecific set of known human HLA molecules. Such HLA sequences can beobtained, e.g., from the IPD-IMGT/HLA database (available atebi.ac.uk/ipd/imgt/hla/) and the international ImMunoGeneTicsInformation System® (available at imgt.org). Nomenclature for such genesis illustrated in FIG. 23. For example, HLA-A1, B8, DR17 is the mostcommon HLA haplotype among Caucasians, with a frequency of 5%. Thus, thedisclosed method can be performed using the known MHC/HLA sequenceinformation in combination with the disclosures provided herein. The HLAsequences are obtainable through online archives or database such asEnsembl (vega.archive.ensembl.org/index.html). As illustrated in FIG.24, the exact location of the HLA-DQA1 gene, the length of therespective gene (exon and intron), and the exact nucleotide sequences ofHLA-DQA1 could be obtained.

In some aspects, the recipient's human leukocyte antigen (HLA) genes andMHC (Class I, II and/or III), are identified and mapped. It will beunderstood that ascertaining the human recipient's HLA/MHC sequence canbe done in any number of ways known in the art. For example, HLA/MHCgenes are usually typed with targeted sequencing methods: eitherlong-read sequencing or long-insert short-read sequencing.Conventionally, HLA types have been determined at 2-digit resolution(e.g., A*01), which approximates the serological antigen groupings. Morerecently, sequence specific oligonucleotide probes (SSOP) method hasbeen used for HLA typing at 4-digit resolution (e.g., A*01:01), whichcan distinguish amino acid differences. Currently, targeted DNAsequencing for HLA typing is the most popular approach for HLA typingover other conventional methods. Since the sequence-based approachdirectly determines both coding and non-coding regions, it can achieveHLA typing at 6-digit (e.g., A*01:01:01) and 8-digit (e.g.,A*01:01:01:01) resolution, respectively. HLA typing at the highestresolution is desirable to distinguish existing HLA alleles from newalleles or null alleles from clinical perspective. Such sequencingtechniques are described in, for example, Elsner H A, Blasczyk R: (2004)Immunogenetics of HLA null alleles: implications for blood stem celltransplantation. Tissue antigens. 64 (6): 687-695; Erlich R L, et al(2011) Next-generation sequencing for HLA typing of Class I loci. BMCgenomics. 12: 42-10.1186/1471-2164-12-42; Szolek A, et al. (2014)OptiType: Precision HLA typing from next-generation sequencing data.Bioinformatics 30:3310-3316; Nariai N, et al. (2015) HLA-VBSeq: AccurateHLA typing at full resolution from whole-genome sequencing data. BMCGenomics 16:S7; Dilthey A T, et al. (2016) High-accuracy HLA typeinference from whole-genome sequencing data using population referencegraphs. PLoS Comput Biol 12:e1005151; Xie C., et al. (2017) Fast andaccurate HLA typing from short-read next-generation sequence data withxHLA 114 (30) 8059-8064, each of which is incorporated herein in itsentirety by reference.

A complete disruption of MHC Class I expression on xenograft has shownto have detrimental effects on the viability of the animal. In a study,SLA Class I expression on porcine cells were abrogated by targeting exon2 of the porcine beta-2-microglobulin gene. The genomic sequencing ofthe produced piglets showed modification at the B2M locus leading to aframeshift, a premature stop codon, and ultimately a functionalknockout. However, the piglets of the study did not survive for morethan 4 weeks due to unexpected disease processes, revealing that suchdisruptive genetic modification may have a negative impact on theviability of the animals. Sake H J, Frenszel A, Lucas-Hahn A, et al.Possible detrimental effects of beta-2-microglobulin knockout in pigs.Xenotransplantation. 2019; 26:e12525.

In one aspect, a replacement template is created for site-directedmutagenic substitutions of nucleotides of the donor swine's SLA/MHCwherein the reprogramming introduces non-transgenic, minimally-requiredalteration that does not result in any frameshifts or frame disruptionsin specific exon regions of the native donor swine's SLA/MHC. Thenucleotide sequence(s) of the replacement template is identified by: a)obtaining a biological sample containing DNA from a transplantrecipient, b) sequencing MHC Class I and II genes in the transplantrecipient's sample, c) comparing the nucleotide sequence of therecipient with that of the donor swine at various loci, and d) creatinga replacement template for one or more of said loci, wherein saidnucleotide sequence of the replacement template are at least 95%identical to the transplant recipient's nucleotide sequences, as furtherdescribed below.

The spreadsheet in FIG. 25A and FIG. 25B, shows human capture referencesequence of exons of DQ-A₁ and DQ-B₁, respectively, of three individualrecipients. As mentioned above, known human HLA/MHC or an individualrecipient's sequenced HLA/MHC sequence(s) may be utilized as a templateto reprogram with precise substitution the swine leukocyte antigen(SLA)/MHC sequence to match, e.g., to have 90%, 95%, 98%, 99%, or 100%sequence homology to a known human HLA/MHC sequence or the humanrecipient's HLA/MHC sequence. As shown in FIG. 25C, the known humanHLA-DQA acquired through online database and individual recipients'sequenced HLA-DQA, can be compared in a nucleotide Sequence Library.FIG. 26D shows comparison of exon 2 region of the swine's SLA-DQAacquired through online database and the known and sequenced recipient'sHLA-DQA1. Both exon 2 region of SLA-DQA and HLA-DQA1 contain 249nucleotides. As illustrated in FIG. 25D, it can be observed that 11% ofthe aligned 249 nucleotides between exon 2 regions of SLA-DQA1 andHLA-DQA1 are completely divergent. Therefore, this disclosure disclosemethod of identifying the non-conserved nucleotide sequences at aspecific exons of human and swine MHC complex. Furthermore, by using ahuman capture reference template, known or sequenced, a site-directedmutagenesis can be performed wherein the specific non-conservednucleotide sequence between the specific exon regions of the SLA geneand the known or recipient's HLA gene are replaced without causing anyframeshift. The site-directed mutagenesis of the SLA-DQA1 and SLA-DQB1gene is shown in FIG. 26A and FIG. 26B, wherein the nucleotide sequencesof the exon 2 region of the recipient specific HLA-DQA1 and HLA-DQB1 areused to create a human capture replacement sequence. Therefore, the useof synthetic replacement template specific to the exon regions of theMHC gene, leads to a non-transgenic, minimally altered genome that doesnot result in any frameshifts or frame disruptions in the native donorswine's SLA/MHC gene.

As mentioned above, disruptive genetic modification that causesframeshifts may have a negative impact on the viability of the animals.Therefore, the present invention discloses method of inhibitingexpression of MHC proteins without causing frameshift in the MHC gene.The spreadsheet in FIG. 25E and FIG. 25F shows human capture referencesequence of exons of DR-A and DR-B₁, respectively, of three individualrecipients. As shown in FIG. 26C and FIG. 26D, by replacing the initialthree nucleotide sequences of the leader exon 1 to a STOP codon, theexpression of DR molecule can be inhibited without causing frameshift.Specifically, for HLA-DRA and DRB₁, the initial three sequences of exon1, ATG, is replaced with stop codon, TAA. Therefore, by using syntheticreplacement template, wherein stop codon is placed in the beginning ofexon 1, the invention provides method of inhibiting expression ofdesired MHC molecule, wherein the non-transgenic, minimally alterationof genome does not result in any frameshifts or frame disruptions in thenative donor swine's SLA/MHC gene.

Further, the beta-2-microglobulin protein which comprises theheterodimer structure of each of the MHC-I proteins is species-specific.Based on the pig genome assembly SSC10.2, a segmental duplication of˜45.5 kb, encoding the entire B2M protein, was identified in pigchromosome 1, wherein functional duplication of the B2M gene identifiedwith a completely identical coding sequence between two copies in pigs.The phylogenetic analysis of B2M duplication in ten mammalian species,confirming the presence of B2M duplication in cetartioldactyls, likecattle, sheep, goats, pigs and whales, but non-cetartiodactyl species,like mice, cats, dogs, horses, and humans. The density of longinterspersed nuclear element (LINE) at the edges of duplicated blocks(39 to 66%) was found to be 2 to 3-fold higher than the average (20.12%)of the pig genome, suggesting its role in the duplication event. The B2MmRNA expression level in pigs was 12.71 and 7.57 times (2−ΔΔCt values)higher than humans and mice, respectively. The identification ofpartially remaining duplicated B2M sequences in the genomes of onlycetartiodactyls indicates that the event was lineage specific. B2Mduplication could be beneficial to the immune system of pigs byincreasing the availability of MHC class I light chain protein, B2M, tocomplex with the proteins encoded by the relatively large number of MHCclass I heavy chain genes in pigs. As shown in FIG. 27, B2M moleculewith respect to MHC Class I molecule can be observed. Further as statedabove and shown in FIG. 27, swine has duplication of B2M gene whilehuman has one. Thus, in one embodiment of the present disclosure, thefirst copy of the swine B2M gene is reprogrammed through site-directedmutagenesis, as previously disclosed. As shown in FIG. 28, the aminoacid sequences of exon 2 of the swine B2M is compared with that of thehuman, wherein the non-conserved regions are identified. In addition,the expression of the second copy of the swine B2M gene is inhibited byuse of STOP codon, as previously disclosed. Thus, in one embodiment ofthe present disclosure includes a genetic modification, wherein thefirst copy of the swine B2M gene is reprogrammed through site-directedmutagenesis and second duplicated B2M gene is not expressed, wherein thereprogramming does not result in frameshift of B2M gene.

Selection and Characterization of Pilot Cell Porcine Line forHumanization by Genetic Modification

Primary macrophages and other antigen presenting cells (APC) are usefulfor studying immune response, however, the long term use of primarycells is limited by the cells' short life span. In addition, primarycells can only be genetically engineered and evaluated one time beforethe cells reach senescence. In the pig model, investigators frequentlyhave used porcine aortic endothelial cells (PAECs) for these type ofstudies. An immortalized cell line that has the desired characteristics(expression of MHC Class I and II molecules and CD80/86) of a macrophageor representative APC would be ideal to conduct multiple modificationsof the genome and address impact on immunological reactivity using thesame genetic background. The ability to generate a viable immortalizedpig cell line has been limited to fibroblasts and epithelial cell lineswhich are not relevant for the study of the immune response inxenotransplantation.

An immortalized porcine alveolar macrophage (PAM) line was developedfrom Landrace strain of pig [Weingartl 2002] and is commerciallyavailable through ATCC [3D4/21, ATCC CRL-2843]. The cell line showedsome percentage of non-specific esterase and phagocytosis which wasdependent upon conditions of the medium. Cells could be grown asanchorage dependent or in colonies under serum free conditions.Myeloid/monocyte markers (e.g. CD14) were detected. Desiredcharacteristics of an immortalized cell line was MHC Class I and II. MHCClass I was shown to be broadly expressed on all cells, however, MHCClass II, DR and DQ, expression of 3D4/21 cells was initially reportedas being low, 18% and 4%. PAEC have been shown to be activated and DRexpression could be upregulated with exposure to IFN-gamma. 3D4/21 cellswere exposed to IFN-gamma and Class II expression increased DR: 29.68%to 42.27% and DQ: 2.28% to 57.36% after 24 hours of exposure toIFN-gamma. In addition, CD80/86 are expressed on the cell surface, theseglycoproteins are essential for the second signal of T cell activationand proliferation. PAM cells, 34D/21, have the desired characteristicsof a porcine APC in which genetic changes in genes associated with theMHC can be documented using an immortalized cell line and the resultingchanges in the phenotype can be assessed using flow cytometry to addressexpression or lack of expression of the glycoproteins of interest andcellular immune responses, Mixed Lymphocyte Response (MLR).

To test for cellular immune response, a one way MLR is set up in whichone set of cells is identified as the stimulator cells, these are donorcells or unmodified or modified PAM cells, and the other set of cells isthe responder cells, these are cells from the recipient (these could befrom recipient's who share a similar expression of MHC molecules are themodified PAM cells. The stimulator cells are treated with an agent toprevent the cells from proliferating and this could be either radiationor incubation with mitomycin C which covalently crosslinks DNA,inhibiting DNA synthesis and cell proliferation. Hence, the stimulatorcells do not proliferate in culture however, the responder cellsproliferate in response to interaction at the MHC Class I and II and itis this proliferation that is measured in a MLR. A cell culturecontaining both stimulator and responder cells is prepared and incubatedfor 5-7 days and proliferation/activation is measured. Proliferation canbe measured by the amount of radioactive thymidine [³HTdr] or BrdU[analog of thymidine] that is incorporated into the DNA uponproliferation at the end of 5 or 7 days.

Combinations of the MLR. Responders cells can be either PBMC, CD4+ Tcells, CD8+ T cells or other subpopulations of T cells. PBMC representall the immune cells that are present in the recipient and the measuredresponse reflects the ability of the responders to mount an immuneresponse to the stimulator cells, [unmodified or modified PAM cells].The measured proliferation consists of both CD4+ and CD8+ T cells whichinteract with MHC Class II and I, respectively. Using only CD4+ T cellsagainst the unmodified or modified PAM cells is to measure the responseto MHC Class II glycoproteins, DR and DQ. To observe a specific responseto DQ, human antigen presenting cells (APCs) are absent from the culturesuch that the cellular response is not the result of pig antigenspresented by the APCs. In parallel, responder CD8+ T cells will be usedto assess an immune response to MHC Class I glycoproteins, SLA 1 AND 2.This type of analysis removes the contribution to the immune responsefrom responder APCs as found in PBMC. Comparative data will demonstratethe contribution of these respective glycoproteins to the immuneresponse of the genetically defined responder and reflects on thegenetic modifications made to the PAM cells.

Flow cytometry, phenotypic analysis of the genetically modified PAMcells. The cell phenotype of genetically modified cells, e.g., cellsfrom a genetically modified animal or cells made ex vivo, are analyzedto measure the changes in expression of the glycoproteins encoded by thegenes that were modified. Cells are incubated with an antibody with afluorescent label that binds to the glycoprotein of interest and labeledcells are analyzed using flow cytometry. The analysis has been performedon unmodified PAM cells to identify the expression of MHC Class I, ClassII (DR and DQ) and CD80/86. Changes in modified PAM cells will bereferenced to this database. Flow cytometry will also be used tocharacterize the expression of glycoproteins encoded by genes for SLA 3,6, 7, and 8 as the genes in the PAM cells are modified with recipientspecific sequences related to HLA C, E, F, and G.

In addition, this type of analysis is also used to ensure theglycoprotein encoded by a gene that is knock-out is not expressed. Thistechnique can also be used to sort out genetically modified cells from apool of cells with mixed phenotypes.

Complement Dependent Cytotoxicity (CDC) assays may be performed todetermine if anti-HLA antibodies recognize the cells from the biologicalproduct of the present disclosure. Assay plates prepared by adding aspecific human serum containing previously characterized anti-HLAantibodies (or control serum) can be used. IFN-γ treated donor cells areresuspended and added to the assay plates, incubated with a source ofcomplement, e.g., rabbit serum. After at least 1 hour of incubation atroom temperature, acridine orange/ethidium bromide solution is added.Percent cytotoxicity is determined by counting dead and live cellsvisualized on a fluorescent microscope, subtracting spontaneous lysisvalues obtained in the absence of anti-HLA antibodies, and scoring witha scale.

NK cell reactivity, modulation to decrease cytotoxicity. Potentialmechanisms of activation, recognition, and elimination of target cellsby NK cells, alone or in combination, induce the release of the contentof their lytic granules (perforin, granzyme, and cytolysin). As anexample, NK cells recognize the lack of self-major histocompatibilitycomplex (MHC) Class I molecules on target cells by inhibitory NK cellreceptors leading to direct NK cytotoxicity. This is the case forxenotransplantation. NK cells are regulated by HLA C that is recognizedby inhibitory NK cell inhibitory killer cell immunoglobulin-likereceptors (KIRs), KIR2DL2/2DL3, KIR2DL1, and KIR3DL1. NK cellsinhibitory receptor, immunoglobulin-like transcript 2 (ILT2) interactswith MHC Class I and CD94-NKG2A recognizing HLA-E. HLA F and G havesimilar roles on the trophoblast. The cytolytic activity of recipient NKcells to an unmodified PAM cell can be measured in vitro in which humanNK cells are added to an adherent monolayer of unmodified PAM cells andcultured for 4 hours. Cell lysis is measured by release of radioactiveCr⁵¹ or a chromophore measured by flow cytometry. PAM cells withmodified SLA 3, 6, 7 or 8 to mirror HLA C, HLA E, HLA G or HLA F,respectively, can be assessed using this cytotoxicity assay.

For knock in cells, the desired sequences are knocked into the cellgenome through insertion of genomic material using, e.g.,homology-directed repair (HDR). To optimize expression of Class IImolecules, the cells are incubated in porcine interferon gamma (IFN-γ)for 72 hours which stimulates expression. Expression is then measured byflow cytometry using target specific antibodies. Flow cytometry mayinclude anti-HLA-C, HLA-E, HLA-G, or other HLA antibodies, or pananti-HLA Class I or Class II antibodies. According to the presentdisclosure, cell surface HLA expression after knock-in is confirmed.

A study was conducted identify the impact of the stimulation by IFN-γand IFN-γ+LPS on the phenotype of the porcine alveolar macrophages (PAM)purchased from ATCC® (3D4/21 cells cat #CRL-2843™) by flow cytometry.

PAM cells were thawed in RPMI-1640/10% FBS and cultured for two days inthree different culture plates. On Day 3, for macrophage activationculture medium was replaced with RPMI-1640/20% FBS medium containing 100ng/mL IFN-γ (Plate 1) and 100 ng/mL IFN-γ plus 10 ng/mL LPS (Plate 2).Untreated cells in RPMI-1640/20% FBS were used as control (Plate 3).Following 24 hours incubation, adherent cells were detached from theplate using TrypLE treatment. Cells were resuspended in FACS buffer(1×PBS pH=7.4, 2 mM EDTA, 0.5% BSA). Cell count and viability weredetermined by trypan blue exclusion method. A total of 1×105 cells werestained with mouse anti pig SLA Class I, SLA Class II DR, SLA Class IIDQ antibodies for 30 min and APC-conjugated CD152(CTLA-4)-muIg fusionprotein (binds to porcine CD80/CD86) for 45 min at 4° C. Cells werewashed two times using FACS buffer and antibody stained cellsresuspended in 100 μL FACS buffer containing anti mouse APC-conjugatedpolyclonal IgG secondary antibody. Followed by incubation for 30 min at4° C. Cells were washed two times using FACS buffer. All cells wereresuspended in 200 μL FACS buffer. Samples were acquired in Novacyteflow cytometry and data was analyzed using NovoExpress.

Analysis procedure is based on NovoExpress flow cytometry analysissoftware. Any equivalent software can be used for the data analysis.Depending on the software used analysis presentation maybe slightlydifferent. Gates maybe named differently and % values might be slightlydifferent.

As shown in FIG. 29, untreated PAM cells result 99.98%, 29.68%, and2.28% SLA Class I, SLA Class II DR and DQ molecules expressionrespectively. These cells were 4.81% CD80/86+. 24 hours of culturingcells in the presence of IFN-γ increased all SLA molecule expression(99.99% SLA Class I+ with increased median fluorescence intensity,42.27% DR+, 57.36% DQ+) and CD80/86 levels (47.38%). IFN-γ containingcells with LPS resulted similar levels of SLA molecules and CD80/86expression compared to cells only treated with IFN-γ.

PAM cells were treated with porcine IFN-γ for 24 hours and stained withprimary MAbs and fluorescein conjugated secondary antibody and APCconjugated CD152 which has a high affinity for co-stimulatory moleculesCD80 (B7-1) and CD86 (B7-2). Upon treatment with IFN-γ, the cellsdisplayed increased SLA and CD80/86 costimulatory molecules expressioncompared to unstimulated PAM cells. While unstimulated cells were 99.98%SLA Class I+, 29.68% DR+2.28 DQ+ and 4.81% CD80/86+, IFN-γ stimulatedcells were 99.99% SLA Class I+, 42.27% DR+, 57.36% DQ+, 47.38% CD80/86+.IFN-γ containing cells with LPS resulted similar levels of SLA moleculesand CD80/86 expression compared to cells only treated with IFN-γ.

In basal conditions, macrophages express low levels of SLA Class II andCD80/86 costimulatory molecules. IFN-γ and IFN-γ-LPS treatment for 24hours induces the expression of SLA Class II and CD80/86 costimulatorymolecules as well as SLA Class I molecules. Extended incubations wouldperhaps increase the expression of these molecules further.

Further, a study was conducted to evaluate the immune proliferativeresponsiveness of human PBMCs (Peripheral Blood Mononuclear Cells), CD8and CD4 positive T cells when they are co-cultured with porcine alveolarmacrophages (PAM) cells. Human donor PBMCs or their CD4+ T cells wereco-cultured with untreated, IFN-γ activated and KLH loaded PAM cells forseven days. As shown in FIG. 30A and FIG. 30B, one-way allogeneic andautologous MLR experiments were performed using the cells isolated fromDonor #11, #50, and #57 as positive and negative controls respectively.Background controls were performed for Mitomycin C (X) treated anduntreated PAM cells, and each human donor cells. Proliferative responseis determined utilizing a bromo-deoxy uridine (BrdU) ELISA assay. On Day6, BrdU addition was completed. On Day 7 media was collected forcytokine (IFN-γ and IL-2) analysis and proliferative responses weredetermined. Cells were observed under the Olympus CK40 microscopy at200× magnification on Day 7 of co-culturing.

As shown in FIG. 31, 72 hours of culturing PAM cells in the presence ofIFN-γ increased SLA Class II DQ molecule expression from 2.55% to95.82%. KLH loaded PAM cells resulted expression of similar level of SLAClass II DQ molecules with untreated cells. All the allogeneic controlshad a positive proliferative response over baseline values and mitomycinC treated PBMCs and PAM cells had a decreased proliferative responsecompared to baseline values. 1×105 purified human CD8+ T cells or humanPBMC were stimulated with increasing numbers of irradiated (30 Gy)porcine PBMC from four-fold knockout pig 10261 or a wild-type pig.Proliferation was measured after 5 d+16 h by 3H-thymidine incorporation.Data representing mean cpm±SEM of triplicate cultures were obtained withcells from one human blood donor in a single experiment. Similarresponse patterns were observed using responder cells from a secondblood donor and stimulator cells from four-fold knockout pig 10262.Proliferation of human CD8+ T cells decreased after stimulation withfour-fold knockout porcine PBMC. (Fischer, et al., 2019). Human PBMCsand CD4+ proliferative responses resulted in allogeneic responses thatwere higher than the xenogeneic responses with PAM cells. Theproliferative responses of three different human CD4+ T cells displayedsimilar xenogeneic responses with PAM cells SI (Stimulation Indexes)values being between 15 and 18.08. The proliferative responses werehighest in xenogeneic cultures from PBMC Donor #57(SI_(w/PAMX, PAM-IFNyX, KLHx)=3.12, 2.75, and 3.79).

Gene Editing Schema to Create Multiple, Independent, Single-VariableHumanized Pilot Porcine Cell Lines by CRISPR-Cas9 Genetic Modification

The genetic modification can be made utilizing known genome editingtechniques, such as zinc-finger nucleases (ZFNs), transcriptionactivator-like effector nucleases (TALENs), adeno-associated virus(AAV)-mediated gene editing, and clustered regular interspacedpalindromic repeat Cas9 (CRISPR-Cas9). These programmable nucleasesenable the targeted generation of DNA double-stranded breaks (DSB),which promote the upregulation of cellular repair mechanisms, resultingin either the error-prone process of non-homologous end joining (NHEJ)or homology-directed repair (HDR), the latter of which is used tointegrate exogenous donor DNA templates. CRISPR-Cas9 may also be used toperform precise modifications of genetic material. For example, thegenetic modification via CRISPR-Cas9 can be performed in a mannerdescribed in Kelton, W. et. al., “Reprogramming MHC specificity byCRISPR-Cas9-assisted cassette exchange,” Nature, Scientific Reports,7:45775 (2017) (“Kelton”), the entire disclosure of which isincorporated herein by reference. Accordingly, the present disclosureincludes reprogramming using CRISPR-Cas9 to mediate rapid and scarlessexchange of entire alleles, e.g., MHC, HLA, SLA, etc.

According to the present disclosure, CRISPR-Cas9 is used to mediaterapid and scarless exchange of entire MHC alleles at specific nativelocus in swine cells. Multiplex targeting of Cas9 with two gRNAs is usedto introduce single or double-stranded breaks flanking the MHC allele,enabling replacement with the template HLA/MHC sequence (provided as asingle or double-stranded DNA template).

In some aspects, the expression of polymorphic protein motifs of thedonor animal's MHC can be further modified by knock-out methods known inthe art. For example, knocking out one or more genes may includedeleting one or more genes from a genome of a non-human animal. Knockingout may also include removing all or a part of a gene sequence from anon-human animal. It is also contemplated that knocking out can includereplacing all or a part of a gene in a genome of a non-human animal withone or more nucleotides. Knocking out one or more genes can also includesubstituting a sequence in one or more genes thereby disruptingexpression of the one or more genes. Knocking out one or more genes canalso include replacing a sequence in one or more genes therebydisrupting expression of the one or more genes without frameshifts orframe disruptions in the native donor swine's SLA/MHC gene. For example,replacing a sequence can generate a stop codon in the beginning of oneor more genes, which can result in a nonfunctional transcript orprotein. For example, if a stop codon is created within one or moregenes, the resulting transcription and/or protein can be disrupted,silenced and rendered nonfunctional.

In another aspect, the present invention utilizes alteration bynucleotide replacement of STOP codon at exon regions of the wild-typeswine's SLA-DR to avoid provocation of natural cellular mediated immuneresponse (CD8+ T Cell) by the recipient, including making cells thatlack functional expression of SLA-DR, SLA-1, SLA-2. For example, thepresent invention utilizes TAA. In other embodiments, the inventionutilizes TAG. In other embodiments, the invention utilizes TGA.

In one aspect, the present invention utilizes insertion or creation (bynucleotide replacement) of STOP codon at exons regions of the wild-typeswine's second, identical duplication B2-microglobulin gene to reducethe B2-microglobulin mRNA expression level in pigs. It will beunderstood that B2-microglobulin is a predominant immunogen,specifically a non-gal xeno-antigen.

In one aspect, the recipient's HLA/MHC gene is sequenced and templateHLA/MHC sequences are prepared based on the recipient's HLA/MHC genes.In another aspect, a known human HLA/MHC genotype from a World HealthOrganization (WHO) database may be used for genetic reprogramming ofswine of the present disclosure.

CRISPR-Cas9 plasmids are prepared, e.g., using polymerase chain reactionand the recipient's HLA/MHC sequences are cloned into the plasmids astemplates. CRISPR cleavage sites at the SLA/MHC locus in the swine cellsare identified and gRNA sequences targeting the cleavage sites and arecloned into one or more CRISPR-Cas9 plasmids. CRISPR-Cas9 plasmids arethen administered into the swine cells and CRIPSR/Cas9 cleavage isperformed at the MHC locus of the swine cells.

The SLA/MHC locus in the swine cells are precisely replaced with one ormore template HLA/MHC sequences matching the known human HLA/MHCsequences or the recipient's sequenced HLA/MHC genes. Cells of the swineare sequenced after performing the SLA/MHC reprogramming steps in orderto determine if the SLA/MHC sequences in the swine cells have beensuccessfully reprogrammed. One or more cells, tissues, and/or organsfrom the HLA/MHC sequence-reprogrammed swine are transplanted into ahuman recipient.

The modification to the donor SLA/MHC to match recipient HLA/MHC causesexpression of specific MHC molecules in the new swine cells that areidentical, or virtually identical, to the MHC molecules of a known humangenotype or the specific human recipient. In one aspect, the presentdisclosure involves making modifications limited to only specificportions of specific SLA regions of the swine's genome to retain aneffective immune profile in the swine while biological products aretolerogenic when transplanted into human recipients such that use ofimmunosuppressants can be reduced or avoided. In contrast to aspects ofthe present disclosure, xenotransplantation studies of the prior artrequired immunosuppressant use to resist rejection. In one aspect, theswine genome is reprogrammed to disrupt, silence, cause nonfunctionalexpression of swine genes corresponding to HLA-A, HLA-B, and DR, and toreprogram via substitution of HLA-C, HLA-E, HLA-F, and/or HLA-G. In someaspects, the swine genome is reprogrammed to knock-out swine genescorresponding to HLA-A, HLA-B, HLA-C, HLA-F, DQ, and DR, and to knock-inHLA-C, HLA-E, HLA-G. In some aspects, the swine genome is reprogrammedto knock-out swine genes corresponding to HLA-A, HLA-B, HLA-C, HLA-F,DQ, and DR, and to knock-in HLA-C, HLA-E, HLA-G, HLA-F, and DQ. In oneaspect, the swine genome is reprogrammed to knock-out SLA-1; SLA-6,7,8;SLA-MIC2; and SLA-DQA; SLA-DQB1; SLA-DQB2, and to knock-in HLA-C; HLA-E;HLA-G; and HLA-DQ. In certain aspects, HLA-C expression is reduced inthe reprogrammed swine genome. By reprogramming the swine cells to beinvisible to a human's immune system, this reprogramming therebyminimizes or even eliminates an immune response that would haveotherwise occurred based on swine MHC molecules otherwise expressed fromthe donor swine cells.

Various cellular marker combinations in swine cells are made and testedto prepare biologically reprogrammed swine cells for acceptance by ahuman patient's body for various uses. For these tests, Porcine AortaEndothelial Cells, fibroblast, or a transformed porcine macrophage cellline available from ATCC® (3D4/21) are used.

The knockout only and knockout plus knock in cell pools are generated bydesigning and synthesizing a guide RNA for the target gene. Each guideRNA is composed of two components, a CRISPR RNA (crRNA) and atrans-activating RNA (tracrRNA). These components may be linked to forma continuous molecule called a single guide RNA (sgRNA) or annealed toform a two-piece guide (cr:tracrRNA).

CRISPR components (gRNA and Cas9) can be delivered to cells in DNA, RNA,or ribonucleoprotein (RNP) complex formats. The DNA format involvescloning gRNA and Cas9 sequences into a plasmid, which is then introducedinto cells. If permanent expression of gRNA and/or Cas9 is desired, thenthe DNA can be inserted into the host cell's genome using a lentivirus.Guide RNAs can be produced either enzymatically (via in vitrotranscription) or synthetically. Synthetic RNAs are typically more purethan IVT-derived RNAs and can be chemically modified to resistdegradation. Cas9 can also be delivered as RNA. The ribonucleoproteins(RNP) format consists of gRNA and Cas9 protein. The RNPs arepre-complexed together and then introduced into cells. This format iseasy to use and has been shown to be highly effective in many celltypes.

After designing and generating the guide RNA, the CRISPR components areintroduced into cells via one of several possible transfection methods,such as lipofection, electroporation, nucleofection, or microinjection.After a guide RNA and Cas9 are introduced into a cell culture, theyproduce a DSB at the target site within some of the cells. The NHEJpathway then repairs the break, potentially inserting or deletingnucleotides (indels) in the process. Because NHEJ may repair the targetsite on each chromosome differently, each cell may have a different setof indels or a combination of indels and unedited sequences.

For knock in cells, the desired sequences are knocked into the cellgenome through insertion of genomic material using, e.g.,homology-directed repair (HDR).

It will be further understood that disruptions and modifications to thegenomes of source animals provided herein can be performed by severalmethods including, but not limited to, through the use of clusteredregularly interspaced short palindromic repeats (“CRISPR”), which can beutilized to create animals having specifically tailored genomes. See,e.g., Niu et al., “Inactivation of porcine endogenous retrovirus in pigsusing CRISPR-Cas-9,” Science 357:1303-1307 (22 Sep. 2017). Such genomemodification can include, but not be limited to, any of the geneticmodifications disclosed herein, and/or any other tailored genomemodifications designed to reduce the bioburden and immunogenicity ofproducts derived from such source animals to minimize immunologicalrejection.

CRISPR/CRISPR-associated protein (Cas), originally known as a microbialadaptive immune system, has been adapted for mammalian gene editingrecently. The CRISPR/Cas system is based on an adaptive immune mechanismin bacteria and archaea to defend the invasion of foreign geneticelements through DNA or RNA interference. Through mammalian codonoptimization, CRISPR/Cas has been adapted for precise DNA/RNA targetingand is highly efficient in mammalian cells and embryos. The mostcommonly used and intensively characterized CRISPR/Cas system for genomeediting is the type II CRISPR system from Streptococcus pyogenes; thissystem uses a combination of Cas9 nuclease and a short guide RNA (gRNA)to target specific DNA sequences for cleavage. A 20-nucleotide gRNAcomplementary to the target DNA that lies immediately 5′ of a PAMsequence (e.g., NGG) directs Cas9 to the target DNA and mediatescleavage of double-stranded DNA to form a DSB. Thus, CRISPR/Cas9 canachieve gene targeting in any N20-NGG site.

Thus, also encompassed by the invention is a genetically modifiednon-human animal whose genome comprises a nucleotide sequence encoding ahuman or humanized MHC I polypeptide and/or β2 microglobulinpolypeptide, wherein the polypeptide(s) comprises conservative aminoacid substitutions of the amino acid sequence(s) described herein.

One skilled in the art would understand that in addition to the nucleicacid residues encoding a human or humanized MHC I polypeptide and/or β2microglobulin described herein, due to the degeneracy of the geneticcode, other nucleic acids may encode the polypeptide(s) of theinvention. Therefore, in addition to a genetically modified non-humananimal that comprises in its genome a nucleotide sequence encoding MHC Iand/or β2 microglobulin polypeptide(s) with conservative amino acidsubstitutions, a non-human animal whose genome comprises a nucleotidesequence(s) that differs from that described herein due to thedegeneracy of the genetic code is also provided.

In an additional or alternative approach, the present disclosureincludes reprogramming, or leveraging the inhibitory and co-stimulatoryeffects of the MHC-I (Class B) molecules. Specifically, the presentdisclosure includes a process that “finds and replaces” portions of thedonor animal genome corresponding to portions of the HLA gene, e.g., tooverexpress HLA-G where possible, retaining and overexpressing portionscorresponding to HLA-E, and/or “finding and replacing” portionscorresponding to HLA-F. As used herein, the term “find and replace”includes identification of the homologous/analogous/orthologousconserved genetic region and replacement of the section or sections withthe corresponding human components through gene editing techniques.

Another aspect includes finding and replacing the beta-2 microglobulinprotein which is expressed in HLA-A, -B, -C, -E, -F, and -G.Homologous/analogous/orthologous conserved cytokine mediating complementinhibiting or otherwise immunomodulatory cell markers, or surfaceproteins, that would enhance the overall immune tolerance atdonor-recipient cellular interface.

In an additional or alternative approach, the present invention utilizesimmunogenomic reprogramming to reduce or eliminate MHC-I (Class A)components to avoid provocation of natural cellular mediated immuneresponse by the recipient. In another aspect, exon regions in the donoranimal (e.g., swine) genome corresponding to exon regions of HLA-A andHLA-B are disrupted, silenced or otherwise nonfunctionally expressed onthe donor animal. In another aspect, exon regions in the donor animal(e.g., swine) genome corresponding to exon regions of HLA-A and HLA-Bare disrupted, silenced or otherwise nonfunctionally expressed in thegenome of the donor animal and exon regions in the donor animal (e.g.,swine) genome corresponding to exon regions of HLA-C may be modulated,e.g., reduced. In one aspect, the present disclosure includes silencing,knocking out, or causing the minimal expression of source animal'sorthologous HLA-C (as compared to how such would be expressed withoutsuch immunogenomic reprogramming).

Further, the beta-2-microglobulin protein which comprises theheterodimer structure of each of the MHC-I proteins is species-specific.Thus, in one embodiment of the present disclosure, it is reprogrammed.In contrast to its counterparts, the genetic instructions encoding forthis prevalent, building-block protein is not located in the MHC-geneloci. Thus, in one embodiment of the present disclosure includes agenetic modification in addition to those specific for the respectivetargets as described herein.

FIG. 33 is a schematic depiction of a humanized porcine cell accordingto the present disclosure. As shown therein, the present disclosureinvolves reprogramming exons encoding specific polypeptides orglycoproteins, reprogramming and upregulating specific polypeptides orglycoproteins, and reprogramming the nuclear genome to havenonfunctional expression of specific polypeptides or glycoproteins, allof which are described in detail herein.

Characterization of Humanized Pilot Porcine Cell Lines and In VitroEvaluation of Resultant Impact to Immunological

Genetically modified cells, e.g., cells from a genetically modifiedanimal or cells made ex vivo, can be analyzed and sorted. In some cases,genetically modified cells can be analyzed and sorted by flow cytometry,e.g., fluorescence-activated cell sorting. For example, geneticallymodified cells expressing a gene of interest can be detected andpurified from other cells using flow cytometry based on a label (e.g., afluorescent label) recognizing the polypeptide encoded by the gene. Inthis application, the surface expression of SLA-1, SLA-2, SLA-3, SLA-6,SLA-7, SLA-8, SLA-DR and SLA-DQ on unmodified PAM cells is establishedusing labeled antibodies directed to epitopes on those glycoproteins. Inthe case of specific gene knock outs (e.g. SLA-1, SLA-2 and SLA-DR),analysis by flow cytometry is used to demonstrate the lack of expressionof these glycoproteins even after incubation of the cells withinterferon gamma. Genes for SLA-3, SLA-6, SLA-7, SLA-8, and SLA-DQ willbe modified such that glycoproteins expressed on the cell surface willreflect HLA-C, HLA-E, HLA-F, HLA-G and HLA-DQ glycoproteins,respectively. Hence a different set of antibodies specific for the HLAepitopes will be used to detect expression of the glycoproteins encodedby the modified genes and antibodies directed to the SLA relatedglycoproteins will not bind to the cell surface of the modified PAMcells.

When knocking out surface sugar glycan epitopes, a cell line that doesnot express the sugar moieties is obtained, so there is no binding ofnatural preformed antibodies found in human serum. This is detectedusing flow cytometry and human serum and a labeled goat anti human IgGor IgM antibody; or specific antibodies directed against sugars; orlabeled sugar specific isolectins. The result is no binding of theantibodies (isolectins) to the final cell line. Positive control is theoriginal cell line (WT) without genetic modifications. In addition, amolecular analysis demonstrates changes in those genes.

For knock in cells, the desired sequences are knocked into the cellgenome through insertion of genomic material using, e.g.,homology-directed repair (HDR). To optimize expression of Class IImolecules, the cells are incubated in porcine interferon gamma (IFN-γ)for up to 72 hours which stimulates expression. Expression is thenmeasured by flow cytometry using target specific antibodies. Flowcytometry may include anti-HLA-C, HLA-E, HLA-G, or other HLA antibodies,or pan anti-HLA Class I or Class II antibodies. According to the presentdisclosure, cell surface HLA expression after knock-in is confirmed.

The immune response of the modified swine cells are evaluated throughMixed Lymphocyte Reaction (MLR) study. Responders cells can be eitherPBMC, CD4+ T cells, CD8+ T cells or other subpopulations of T cells.PBMC represent all the immune cells that are present in the recipientand the measured response reflects the ability of the responders tomount an immune response to the stimulator cells, for example, acomparison of unmodified PAM cells and modified PAM cells.Alternatively, PAECs or fibroblasts may be used. The measuredproliferation consists of both CD4+ and CD8+ T cells which interact withMHC Class II and I, respectively. Using only CD4+ T cells against theunmodified or modified PAM cells measures the response to MHC Class IIglycoproteins, DR and DQ. For example, in an MLR where SLA DR is knockedout in the PAM cells, the CD4+ T cell proliferative response will bedecreased; or when SLA-DQ gene is modified by using a sequence from a“recipient” [the responder] the proliferative response will be decreasedsince in this case the responder recognizes the DQ glycoprotein as self,whereas, in the DR knock-out, DR was absent and thus a signal could notbe generated.

Responder CD8+ T cells were used to assess an immune response to MHCClass I glycoproteins, SLA-1 and SLA-2. 1×10⁵ purified human CD8+ Tcells (A) or human PBMC (B) were stimulated with increasing numbers ofirradiated (30 Gy) porcine PBMC from four-fold knockout pig 10261 or awild-type pig. Proliferation was measured after 5 d+16 h by 3H-thymidineincorporation. Data represent mean cpm±SEM of triplicate culturesobtained with cells from one human blood donor in a single experiment.Similar response patterns were observed using responder cells from asecond blood donor and stimulator cells from four-fold knockout pig10262. Proliferation of human CD8+ T cells decreased after stimulationwith four-fold knockout porcine PBMC. (Fischer, et al., Viable pigsafter simultaneous inactivation of porcine MHC Class I and threexenoreactive antigen genes GGTA1, CMAH and B4GALNT2,Xenotransplantation, 2019). Modified knock out PAM cells not expressingSLA-1 and SLA-2 will not generate a CD8+ T cell response. This is incontrast with a response using PBMC as the responders. See FIG. 34.

Complement Dependent Cytotoxicity (CDC) assays may be performed todetermine if anti-HLA antibodies recognize the cells from the biologicalproduct of the present disclosure. Assay plates prepared by adding aspecific human plasma containing previously characterized anti-HLAantibodies (or control plasma) can be used. Plasma is serially dilutedstarting at 1:50 to 1:36450 in HBSS media with calcium and magnesium,incubated with modified or unmodified PAM cells for 30 minutes at 4° C.followed by incubation with freshly reconstituted baby rabbit complementfor 1 hour at 37° C. The cells were then stained with FluoresceinDiacetate (FDA) and Propidium Iodide (PI) for 15 minutes and analyzed byflow cytometry. Appropriate compensation controls were run for eachassay. Cells were acquired and analyzed on an ACEA NovoCyte FlowCytometer. PAM cells can also be treated with interferon gamma toincrease surface expression of MHC molecules.

Cell populations were determined for the following conditions:

-   -   a. Dead Cells: PI+, FDA−    -   b. Damaged Cells: PI+, FDA+    -   c. Live Cells: PI−, FDA+

Appropriate calculations were performed to determine % cytotoxicity foreach concentration (dilution) of plasma, and the results plotted inPrism. Based on the cytotoxicity curve, the required dilution for 50%kill (IC50) was determined. This is illustrated using human plasmaagainst WT or GalTKO porcine PBMC in FIG. 36A and FIG. 36B, wherereduced cytotoxicity was identified against cells lacking a1,3-galactose on the glycoproteins.

NK cytotoxicity against unmodified and modified PAM cells where genesfor SLA 3, SLA 6, SLA 7, and SLA 8 are modified such that glycoproteinsexpressed on the cell surface will reflect HLA C, HLA E, HLA F, and HLAG glycoproteins, respectively. The cytotoxic activity of freshlyisolated and IL-2-activated human NK cells was tested in 4-hr 51Crrelease assays in serum-free AIM-V medium. Labeled unmodified andmodified PAM cells are cultured in triplicate with serial 2-folddilutions of NK cells four E:T ratios ranging from 40:1 to 5:1. Afterincubation for 4 hr at 37° C., the assays are stopped, ⁵¹Cr release isanalyzed on a gamma counter, and the percentage of specific lysis wascalculated. NK cells from a specific genetically matched “recipient”will have reduced killing of modified PAM cells compared to unmodifiedPAM cells. The protection provided by HLA E in transfected PAEC cellsagainst NK cells is illustrated in FIG. 34.

HLA E expression on porcine lymphoblastoid cells inhibits xenogeneichuman NK cytotoxicity. NK cytotoxicity of 2 donors, KH and MS, against13271-E/A2 or 13271-E/B7 (solid diamonds) transfected with HLA E/A2 orHLA E/B7, respectively or untransfected 13271 cells (open triangle). Tooptimize expression of Class II molecules, the cells are incubated inporcine interferon gamma (IFN-γ) for 72 hours which stimulatesexpression. Expression is then measured by flow cytometry using targetspecific antibodies. Flow cytometry may include anti-HLA-C, HLA-E,HLA-G, or other HLA antibodies, or pan anti-HLA Class I or Class IIantibodies. According to the present disclosure, cell surface HLAexpression after knock-in is confirmed.

Multiple, Simultaneous Genetic Modifications in a Single Pilot PorcineCell Line to Achieve Relative Humanized Phenotype and ConsequentialReduction of CD8+, CD4+, and Natural Killer Cell Immune Reactivity as aDirect Result of Multiple CRISPRCas9 Genetic Modification Schema

In some aspects, genetic modifications in a porcine cell line to insertthe modifications listed in table listed in FIG. 33. In some aspects, inaddition to the genetic modifications listed in FIG. 33, the threepredominant swine cell surface glycans (alpha-Gal, Neu5Gc, and Sda) arenot expressed in order to reduce the hyperacute rejection phenomenon andthe deleterious activation of antibody-mediated immune pathways, namelyactivation of the complement cascade. With this step, creation of anallogeneic-“like” cell with respect to non-MHC cell markers is grosslyachieved.

Genetically modified cells, e.g., cells from a genetically modifiedanimal or cells made ex vivo, are analyzed and sorted. In some cases,genetically modified cells can be analyzed and sorted by flow cytometry,e.g., fluorescence-activated cell sorting. For example, geneticallymodified cells expressing a gene of interest can be detected andpurified from other cells using flow cytometry based on a label (e.g., afluorescent label) recognizing the polypeptide encoded by the gene. Thegene of interest may be as small as a few hundred pairs of cDNA bases,or as large as about a hundred thousand pairs of bases of a genic locuscomprising the exonic-intron encoding sequence and regulation sequencesnecessary to obtain an expression controlled in space and time.Preferably, the size of the recombined DNA segment is between 25 kb andlonger than 500 kb. In any case, recombined DNA segments can be smallerthan 25 kb and longer than 500 kb.

It will be further understood that causing the donor swine cells,tissues, and organs to express a known human MHC genotype or therecipient's MHC specifically as described herein, combined with theelimination in the donor swine cells of alpha-1,3-galactosytransferase,Neu5Gc, and β1,4-N-acetylgalactosaminyltransferase (B4GALNT2) (e.g.,“single knockout,” “double knockout,” or “triple knockout”), presents aswine whose cells will have a decreased immunological rejection ascompared to a triple knockout swine that lacks the specific SLA/MHCreprogramming of the present disclosure.

The immune response of the modified swine cells are evaluated throughMixed Lymphocyte Reaction (MLR) study. The impact of the modification ornon-expression of MHC Ia polypeptides on the immune response aremeasured through the immune response of CD8+ T Cells. The impact of themodification of MHC Ib polypeptides on the immune response are measuredthrough the immune response of NK Cells. The impact of the modificationor non-expression of MHC II polypeptides on the immune response aremeasured through the immune response of CD4+ T Cells. The MLR study,herein, not only measures the efficacy of the site-directed mutagenicsubstitution, but also evaluates and identifies the impact of individualmodifications, individually and as a whole, as measurements are takeniteratively as additional site-directed mutagenic substitutions aremade.

For knock in cells, the desired sequences are knocked into the cellgenome through insertion of genomic material using, e.g.,homology-directed repair (HDR). To optimize expression of Class IImolecules, the cells are incubated in porcine interferon gamma (IFN-γ)for 72 hours which stimulates expression. Expression is then measured byflow cytometry using target specific antibodies. Flow cytometry mayinclude anti-HLA-C, HLA-E, HLA-G, or other HLA antibodies, or pananti-HLA Class I or Class II antibodies. According to the presentdisclosure, cell surface HLA expression after knock-in is confirmed.

Complement Dependent Cytotoxicity (CDC) assays may be performed todetermine if anti-HLA antibodies recognize the cells from the biologicalproduct of the present disclosure. Assay plates prepared by adding aspecific human serum containing previously characterized anti-HLAantibodies (or control serum) can be used. IFN-γ treated donor cells areresuspended and added to the assay plates, incubated with a source ofcomplement, e.g., rabbit serum. After at least 1 hour of incubation atroom temperature, acridine orange/ethidium bromide solution is added.Percent cytotoxicity is determined by counting dead and live cellsvisualized on a fluorescent microscope, subtracting spontaneous lysisvalues obtained in the absence of anti-HLA antibodies, and scoring witha scale.

When knocking out or otherwise silencing surface sugar glycans, a cellline that does not express the sugar moieties is obtained, so there isno binding of natural preformed antibodies found in human serum. This isdetected using flow cytometry and human serum and a labeled goat antihuman IgG or IgM antibody; or specific antibodies directed againstsugars. The result is no binding of the antibodies to the final cellline. Positive control is the original cell line (WT) without geneticmodifications. In addition, a molecular analysis demonstrates changes inthose genes.

In knocking out or otherwise silencing expression of SLA Class Imolecules using CRISPR technologies, the resulting cell line lacks theabove sugar moieties as well as SLA Class I expression. Analysis by flowcytometry and molecular gene are performed to demonstrate no surfaceexpression and changes made at the gene level. Cellular reactivity isassessed using a mixed lymphocyte reaction (MLR) with human PBMCs andthe irradiated cell line. In comparison to the WT line, there is areduction in the T cell proliferation, predominantly in the CD8+ Tcells.

Reactivity against expression of SLA Class II molecules, DR and DQ isalso minimized or eliminated (there is no porcine DP). Analysis isperformed at the molecular level, cell surface expression, and in vitroreactivity with human PBMC. There is a significant downward modulationof reactivity against the resulting cell line.

To test for cellular reactivity, all cells are incubated with porcineIFN-γ for 72 hours then human CD4+ T cells are added to porcine celllines and cultured for 7 days. The readout is a form ofactivation/proliferation depending on the resources available.

To observe a specific response to DQ, human antigen presenting cells(APCs) are absent from the culture such that the cellular response isnot the result of pig antigens presented by the APCs.

Creation of a Humanized, “Bespoke”, Designated-Pathogen Free,(Non-Human) Donor of Cells, Tissues, and Organs for Transplantation

Others have attempted to develop homozygous transgenic pigs, which is aslow process, requiring as long as three years using traditional methodsof homologous recombination in fetal fibroblasts followed by somaticcell nuclear transfer (SCNT), and then breeding of heterozygoustransgenic animals to yield a homozygous transgenic pig. The attempts atdeveloping those transgenic pigs for xenotransplantation has beenhampered by the lack of pluripotent stem cells, relying instead on thefetal fibroblast as the cell upon which genetic engineering was carriedout. For instance, the production of the first live pigs lacking anyfunctional expression of α(1,3) galactosyltransferase (GTKO) was firstreported in 2000. In contrast to such prior attempts, the presentdisclosure provides a faster and fundamentally different process formaking non-transgenic reprogrammed swine as disclosed herein. In someaspects, porcine fetal fibroblast cells are reprogrammed using geneediting, e.g., by using CRISPR/Cas for precise reprogramming andtransferring a nucleus of the genetically modified porcine fetalfibroblast cell to a porcine enucleated oocyte to generate an embryo;and d) transferring the embryo into a surrogate pig and growing thetransferred embryo to the genetically modified pig in the surrogate pig.

Upon confirmation of study results, genetically reprogrammed pigs arebred so that several populations of pigs are bred, each populationhaving one of the desirable human cellular modifications determined fromthe above assays. The pigs' cellular activity after full growth isstudied to determine if the pig expresses the desired traits to avoidrejection of the pigs' cells and tissues after xenotransplantation.Thereafter, further genetically reprogrammed pigs are bred having morethan one of the desirable human cellular modifications to obtain pigsexpressing cells and tissues that will not be rejected by the humanpatient's body after xenotransplantation.

The generation of an induced pluripotent stem cell (iPSC) from pigsoffers an opportunity beyond the use of primary cells from fetalfibroblasts. The ability of iPSC to proliferate almost indefinitely,which contrasts with the limited number of cell divisions that primarysomatic cells can undergo before they senesce, likely means that theiPSC will tolerate the multiple selection steps needed to accommodatedirected changes in several genes, especially for gene knock-outs andknock-ins, before nuclear transfer. Another advantage of iPSC oversomatic cells is that it has been predicted that cloning efficiencyshould be inversely correlated with differentiation state and associatedepigenetic state. The PAM cells presented in this disclosure are atransformed cell line but the genetic engineering schema can betransferred to porcine iPSC. The specific genetically modified iPSC linewould then be used for somatic cell nuclear transfer (SCNT),transferring a nucleus of the genetically modified porcine fetalfibroblast cell to a porcine enucleated oocyte to generate an embryo;and transferring the embryo into a surrogate pig and growing thetransferred embryo to the genetically modified pig in the surrogate pig.This has the advantage in that the transferred nucleus contains thespecific genome, hence the piglets do not need to go through breeding toobtain a homozygous offspring. The genotype and phenotype of the pigletsare identical to the iPSC.

Specific populations of gene modified iPSC can be cryopreserved as aspecific cell line and used as required for development of pigs neededfor that genetic background. Thawed iPSCs are cultured and nucleus istransferred into enucleated oocytes to generate blastocysts/embryos forimplantation into surrogate pig. This creates a viable bank ofgenetically modified iPSC for generation of pigs required for patientspecific tissue, organ, or cell transplantation.

Restated, the former/previous approach to this unmet clinical need hasprecisely followed the classic medical dogma of “one-size fits all”.Instead of following this limited approach, we pragmatically demonstratethe ability to harness present technological advances and fundamentalprinciples to achieve a “patient-specific” solution which dramaticallyimproves clinical outcome measures. The former, we refer as the“downstream” approach—which must contend with addressing all of thenatural immune processes in sequence. The latter, our approach, weoptimistically term the “upstream” approach—one which represents theculmination of unfilled scientific effort into a coordinatedtranslational effort.

In another aspect, disclosed herein is a method for making a geneticallymodified animal described in the application, comprising: a) obtaining acell with reduced expression of one or more of a component of a MHCI-specific enhanceosome, a transporter of a MHC I-binding peptide,and/or C3; b) generating an embryo from the cell; and c) growing theembryo into the genetically modified animal. In some cases, the cell isa zygote.

In certain aspects, HLA/MHC sequence-reprogrammed swine are bred for atleast one generation, or at least two generations, before their use as asource for live tissues, organs and/or cells used inxenotransplantation. In certain aspects, the CRISPR/Cas9 components canalso be utilized to inactivate genes responsible for PERV activity,e.g., the pol gene, thereby simultaneously completely eliminating PERVfrom the swine donors.

In certain aspects, the present disclosure includes embryogenesis andlive birth of SLA-free and HLA-expressing biologically reprogrammedswine. In certain aspects, the present disclosure includes breedingSLA-free and HLA-expressing biologically reprogrammed swine to createSLA-free and HLA-expressing progeny. In certain aspects, the CRISPR/Cas9components are injected into swine zygotes by intracytoplasmicmicroinjection of porcine zygotes. In certain aspects, the CRISPR/Cas9components are injected into swine prior to selective breeding of theCRISPR/Cas9 genetically modified swine. In certain aspects, theCRISPR/Cas9 components are injected into donor swine prior to harvestingcells, tissues, zygotes, and/or organs from the swine. In certainaspects, the CRISPR/Cas9 components include all necessary components forcontrolled gene editing including self-inactivation utilizing governinggRNA molecules as described in U.S. Pat. No. 9,834,791 (Zhang), which isincorporated herein by reference in its entirety.

Upon confirmation of study results, genetically reprogrammed pigs arebred so that several populations of pigs are bred, each populationhaving one of the desirable human cellular modifications determined fromthe above assays. The pigs' cellular activity after full growth isstudied to determine if the pig expresses the desired traits to avoidrejection of the pigs' cells and tissues after xenotransplantation.Thereafter, further genetically reprogrammed pigs are bred having morethan one of the desirable human cellular modifications to obtain pigsexpressing cells and tissues that will not be rejected by the humanpatient's body after xenotransplantation.

Any of the above protocols or similar variants thereof can be describedin various documentation associated with a medical product. Thisdocumentation can include, without limitation, protocols, statisticalanalysis plans, investigator brochures, clinical guidelines, medicationguides, risk evaluation and mediation programs, prescribing informationand other documentation that may be associated with a pharmaceuticalproduct. It is specifically contemplated that such documentation may bephysically packaged with cells, tissues, reagents, devices, and/orgenetic material as a kit, as may be beneficial or as set forth byregulatory authorities.

In another aspect, disclosed herein is a method for making a geneticallymodified animal described in the application, comprising: a) obtaining acell with reduced expression of one or more of a component of a MHCI-specific enhanceosome, a transporter of a MHC I-binding peptide,and/or C3; b) generating an embryo from the cell; and c) growing theembryo into the genetically modified animal. In some cases, the cell isa zygote.

Muscle and skin tissue samples taken from each of these pigs weredissected and cultured to grow out the fibroblast cells. The cells werethen harvested and used for somatic cell nuclear transfer (SCNT) toproduce clones. Multiple fetuses (up to 8) were harvested on day 30.Fetuses were separately dissected and plated on 150 mm dishes to growout the fetal fibroblast cells. Throughout culture, fetus cell lineswere kept separate and labeled with the fetus number on each tube orculture vessel. When confluent, cells were harvested and frozen at about1 million cells/mL in FBS with 10% DMSO for liquid nitrogencryo-storage.

Added from different example: In certain aspects, the CRISPR/Cas9components are injected into swine oocytes, ova, zygotes, or blastocytesprior to transfer into foster mothers.

Creation of, Procurement of Personalized, Tolerogenic Cells, Tissues,and Organs Donor of Cells, Tissues, and Organs for Transplantation fromHumanized, “Bespoke”, Designated-Pathogen Free, (Non-Human) Donor SourceAnimal Facility (“SAF”)

Referring to FIG. 37, a barrier source animal location, including, butnot limited to, a Source Animal Facility (“SAF”) 100, that can be usedfor the housing, propagation, maintenance, care and utilization of aclosed colony swine, including a closed colony that is designatedpathogen free (“DPF”) (“DPF Closed Colony”) 102, is shown. As containedherein, the SAF has positive pressure, biocontainment characteristics isoperated under specific isolation-barrier conditions.

As described herein, the DPF Closed Colony 102 is comprised of sourceanimals maintained and propagated for harvesting various biologicalproducts for use in human xenotransplantation and other therapies,wherein such products have reduced bioburden and demonstrate reducedimmunogenicity resulting from xenotransplantation and other therapeuticprocedures. In some aspects, xenotransplantation products of the presentdisclosure are less immunogenic than a xenotransplantation product madefrom conventional Gal-T knockout swine, from conventional tripleknockout swine, from transgenic swine, from wild-type animals, and/orallograft. For example, as shown in Examples 1 and 2, biologicalproducts made according to the present disclosure provided unexpectedlyhigh clinical benefit when using a single knockout pig as the donoranimal in that, despite the presence of Neu5Gc and porcine B4GALNT2, thebiological product made according to the present disclosure had lessimmunogenicity than allograft, vascularized, and was resistant torejection for the entire duration of the study period.

As further described herein, the SAF 100 and each of its accompanyingareas (e.g., rooms, suites or other areas) can be utilized to house andmaintain source animals from which biological products are harvestedand/or processed. The SAF 100 and its areas are designed to minimize andeliminate the potential for contamination of the harvested and/orprocessed biological products and cross-contamination between suchproducts.

Within the SAF 100, in some aspects, utilized animal areas areventilated. For example, animal areas are ventilated with highefficiency particulate air (HEPA)-filtered fresh air from the roof ofthe building, for example, having at least 10-15 air changes per hour.Additionally, one or more laminar flow hoods (e.g., Class II Type A2Laminar Airflow Biosafety Cabinets) are utilized in the SAF rooms,including in a xenotransplantation drug processing suite to providingadditional ventilation to minimize or eliminate cross contamination.

In some aspects, utilized areas are also temperature controlled andmonitored. For example, the areas are heated and cooled to maintaintemperature within the range specified by, for example, the Guide forthe Care and Use of Laboratory Animals. Utilized animal holding roomsare also alarmed and centrally monitored for high or low temperatures,and staff are notified immediately if temperatures are beyond requiredtemperature.

In some aspects, the SAF 100 has multiple levels of containment for thesource animals. For example, source animals are contained in a primarylevel of containment consisting of pens and cages which are secured bystainless steel latches. With respect to secondary level of containment,functionally designated areas (e.g., rooms, suites or other areas) canhave latched inner doors, and an ante-room with card-controlled accessto a hallway. A tertiary level of containment can include outsideperimeter fencing.

The entire SAF is located within a single building. Primary entrance isthrough a single door via programmable identification (ID) card. Allother external doors are alarmed, remain locked, and are for emergencyuse only.

Security is also a consideration to ensure security of the SAF 100 ingeneral, and to control individuals entering the SAF 100 to minimize therisk of outside contaminants entering the SAF 100 and reaching thesource animals. Therefore, in one aspect, the primary entrance to theSAF 100 is through a single door 116 via programmable identification(ID) card 118. All other external doors 120 are alarmed, remain locked,and are for emergency use only.

It will be understood that the SAF 100 and its features as disclosedherein are set out as examples, and it will be further understood thatother facilities with various features can also be utilized to performthe methods and produce the products disclosed herein.

In some aspects, the SAF 100 animal program is licensed and/oraccredited and overseen, evaluated and operated by a team of highlyexperienced, professional staff. For example, the program is registeredand/or accredited with the USDA Animal and Plant Health InspectionService (as a licensed animal research facility), National Institute ofHealth (NIH) Office of Laboratory Animal Welfare (OLAW) (confirmingcompliance with Public Health and Safety (PHS) regulations, Associationfor Assessment and Accreditation of Laboratory Animal Care (AAALAC)(with veterinary care of the source animals housed at the SAF under thedirection of an attending veterinarian), and other federal, state andlocal regulatory authorities.

In some aspects, to ensure the welfare of the source animals, SAFpersonnel, and caretakers of source animals adhere to procedures foranimal husbandry, tissue harvesting, and termination of animals that areapproved by an appropriate Institutional Animal Care and Use Committee,in accordance with the Animal Welfare Act (7 U.S.C. 2131, et seq.),accredited by the AAALAC, and in compliance of the standards as setforth in the Guide for the Care and Use of Laboratory Animals.

In some aspects, caretakers have extensive training and experience inhandling and caring for the source animals being managed in accordancewith the present invention. For example, each caretaker undergoes adocumented training program covering the standard operating proceduresgoverning handling and care of these source animals, and be skilled inmaking daily health assessments and insuring prompt care is directed toany animal in need. In addition, the caretakers can be trained inscrubbing and gowning procedures prior to entry into the isolation areas(e.g., rooms, suites or other areas) as described herein, and under amedical surveillance program to ensure staff health and the health ofthe source animals.

To minimize and eliminate contamination risk to the SAF, any personnelor visitors entering the SAF wear personnel protective equipment orchange into facility dedicated clothing and footwear before entry intoany containment areas. Visitors who wish to enter animal areas must nothave had any contact with live swine for at least 24 hours preceding thevisit or must shower at the facility prior to entry.

It will be understood that the approaches and procedures set forthherein are examples as to how to ensure contamination does not reach thesource animals within SAF 100. It will be further understood that amultitude of approaches can also be utilized to achieve a designatedpathogen free environment for source animals.

Source Animals

In some aspects, as described herein, swine can be utilized as sourceanimals. As used herein, unless otherwise specified, the terms “swine,”“pig” and “porcine” are generic terms referring to the same type ofanimal without regard to gender, size, or breed. It will be understoodthat any number of source animals could be utilized in accordance withthe present invention, including, but not limited to, pigs, non-humanprimates, monkeys, sheep, goats, mice, cattle, deer, horses, dogs, cats,rats, mules, and any other mammals. Source animals could also includeany other animals including, but not limited to, birds, fish, reptiles,and amphibians.

It will be further understood that any animal serving as a source animalhereunder, including swine, regardless of how such swine may beconfigured, engineered, or otherwise altered and/or maintained, may becreated, bred, propagated and/or maintained in accordance with thepresent disclosure to create and maintain animals and resultingbiological products to be used in or in preparation or pursuit ofclinical xenotransplantation.

For example, the present disclosure includes non-human animals, e.g.,swine, having certain combinations of specific genetic characteristics,breeding characteristics and pathogen-free profile. Such animals mayinclude, as described above and herein, immunogenomic reprogrammed swinehaving a biologically reprogrammed genome such that it does not expressone or more extracellular surface glycan epitopes, e.g., genes encodingalpha-1,3 galactosyltransferase, cytidinemonophosphate-N-acetylneuraminic acid hydroxylase (CMAH), andβ1,4-N-acetylgalactosaminyltransferase are disrupted such that surfaceglycan epitopes encoded by said genes are not expressed, as well asother modifications to the swine's SLA to express MHC-I or MHC-II, andregulation of PD-1 and CTLA4, as described above and herein. Resultingfrom the process described herein, the swine is free of at least thefollowing zoonotic pathogens:

-   -   (i) Ascaris species, Cryptosporidium species, Echinococcus,        Strongyloids sterocolis, and Toxoplasma gondii in fecal matter;    -   (ii) Leptospira species, Mycoplasma hyopneumoniae, porcine        reproductive and respiratory syndrome virus (PRRSV),        pseudorabies, transmissible gastroenteritis virus (TGE)/Procine        Respiratory Coronavirus, Toxoplasma Gondii in antibody titers;    -   (iii) Porcine Influenza;    -   (iv) the following bacterial pathogens as determined by        bacterial culture: Bordetella bronchisceptica,        Coagulase-positive staphylococci, Coagulase-negative        staphylococci, Livestock-associated methicillin resistant        Staphylococcus aureus (LA MRSA), Microphyton and Trichophyton        spp.;    -   (v) Porcine cytomegalovirus; and    -   (vi) Brucella suis;    -   is raised and maintained according to a bioburden-reducing        procedure, the procedure comprising maintaining the swine in an        isolated closed herd, wherein all other animals in the isolated        closed herd are confirmed to be free of said zoonotic pathogens;        wherein the swine is isolated from contact with any non-human        animals and animal housing facilities outside of the isolated        closed herd.

As indicated previously, in some aspects, the swine source animals mayhave a combination of one or more genetic modifications including“knockout” and/or “knock-in” swine having one or more characteristics ofswine disclosed in U.S. Pat. No. 7,795,493 (“Phelps”), the entiredisclosure of which is incorporated herein by reference. Such swine lackactive (and/or have disrupted) α-(1,3) galactosyl epitopes responsiblefor hyperacute rejection in humans upon transplantation. Multiplemethods of production of knockout/knock-in swine are disclosed in Phelpsincluding: the inactivation of one or both alleles of the alpha-1,3-GTgene by one or more point mutations (for example by a T-to-G pointmutation at the second base of exon 9) and/or genetic targeting eventsas disclosed at col. 9, line 6 to col. 10, line 13; col. 21, line 53 tocol. 28, line 47; and col. 31, line 48 to col. 38, line 22 of Phelps,incorporated herein by reference. The creation of such swine through thedescribed methods, and/or the utilization of such swine and progenyfollowing creation, can be employed in the practice of the presentinvention, including, but not limited to, utilizing organs, tissueand/or cells derived from such swine.

Similarly, in other aspects, the swine source animals include “knockout”and “knock-in” swine having one or more characteristics of swinedisclosed in U.S. Pat. No. 7,547,816 (“Day”), the entire disclosure ofwhich is incorporated herein by reference. Such swine also lack active(and/or have disrupted) α-(1,3) galactosyl epitopes responsible forhyper-acute rejection in humans upon transplantation. Multiple methodsof production of knockout/knock-in swine are disclosed in Day including:enucleating an oocyte, fusing the oocyte with a porcine cell having anon-functional alpha-1,3-GT gene, followed by implantation into asurrogate mother, as described more fully at col. 4, line 61 to col. 18,line 55 of Day, incorporated herein by reference. The creation of suchswine through the described methods, and/or the utilization of suchswine and progeny following creation, can be employed in the practice ofthe present invention, including, but not limited to, utilizing organs,tissue and/or cells derived from such swine.

Similarly, in other aspects, the swine source animals include GGTA Null(“knockouts” and “knock-ins”) swine having one or more characteristicsof swine disclosed in U.S. Pat. No. 7,547,522 (“Hawley”), the entiredisclosure of which is incorporated herein by reference. Such swine alsolack active (and/or have disrupted) α-(1,3) galactosyl epitopesresponsible for hyper-acute rejection in humans upon transplantation. Asdisclosed in Hawley, production of knockout/knock-in swine includesutilizing homologous recombination techniques, and enucleating oocytesfollowed by fusion with a cell having a non-functional alpha-1,3-GT geneand implantation into a surrogate mother (as disclosed more fully atcol. 6, line 1 to col. 14, line 31). The creation of such swine throughthe described methods, and/or the utilization of such swine and progenyfollowing creation, can be employed in the practice of the presentinvention, including, but not limited to, utilizing organs, tissueand/or cells derived from such swine.

In yet other aspects, the swine source animals include swine and swinethat lack active (and/or have disrupted) α-(1,3) galactosyl epitopeshaving one or more characteristics of swine as described in U.S. Pat.No. 9,883,939 (“Yamada”), the entire disclosure of which is incorporatedby reference herein. In certain aspects, the swine source animals foruse or modification in accordance with the present disclosure includethe swine having one or more characteristics of swine described in U.S.2018/0184630 (Tector, III), the disclosure of which is incorporated byreference herein in its entirety. The creation of such swine through thedescribed methods, and/or the utilization of such swine and progenyfollowing creation, can be employed in the practice of the presentinvention, including, but not limited to, utilizing organs, tissueand/or cells derived from such swine.

In yet other aspects, swine source animals include the swine having oneor more characteristics of swine disclosed in U.S. Pat. No. 8,106,251(Ayares), U.S. Pat. No. 6,469,229 (Sachs), U.S. Pat. No. 7,141,716(Sachs), each of the disclosures of which are incorporated by referenceherein. The creation of such swine through the described methods, and/orthe utilization of such swine and progeny following creation, can beemployed in the practice of the present invention, including, but notlimited to, utilizing organs, tissue and/or cells derived from suchswine.

In some aspects, the swine can originate from one or more highly inbredherds of pigs (whether genetically modified or not (i.e., wild-type))with a co-efficient of inbreeding of 0.50 or greater. A highercoefficient of inbreeding indicates the products derived from the sourceanimals may have more consistent biological properties for use inpig-to-human xenotransplantation (e.g., a coefficient of inbreeding of0.80 or greater in one aspect). Coefficients of inbreeding for animalsare disclosed in Mezrich et al., “Histocompatible Miniature Swine: AnInbred Large-Animal Model,” Transplantation, 75(6):904-907 (2003). Anexample of a highly inbred herd of swine includes miniature swinedescendant from the miniature swine disclosed in Sachs, et al.,“Transplantation in Miniature Swine. I. Fixation of the MajorHistocompatibility Complex,” Transplantation 22:559 (1976), which is ahighly inbred line possessing reasonable size matches particularly fororgans eventually utilized for clinical transplantation. The creation ofsuch swine through the described methods, and/or the utilization of suchswine and progeny following creation, can be employed in the practice ofthe present invention, including, but not limited to, utilizing organs,tissue and/or cells derived from such swine.

Source animals can also include animals swine that lack active (and/orhave disrupted) alpha-1,3-galactosyltransferase, Neu5Gc, andβ1,4-N-acetylgalactosaminyltransferase as described in U.S. PatentPublication No. US2017/0311579 (Tector), the entire disclosure of whichis incorporated herein by reference. The creation of such swine throughthe described methods, and/or the utilization of such swine and progenyfollowing creation, can be employed in the practice of the presentinvention, including, but not limited to, utilizing organs, tissueand/or cells derived from such swine.

It is therefore understood that multiple source animals, with an arrayof biological properties including, but not limited to, genomemodification and/or other genetically engineered properties, can beutilized to reduce immunogenicity and/or immunological rejection (e.g.,acute, hyperacute, and chronic rejections) in humans resulting fromxenotransplantation. In certain aspects, the present disclosure can beused to reduce or avoid thrombotic microangiopathy by transplanting thebiological product of the present disclosure into a human patient. Incertain aspects, the present disclosure can be used to reduce or avoidglomerulopathy by transplanting the biological product of the presentdisclosure into a human patient. It will be further understood that thelisting of source animals set forth herein is not limiting, and thepresent invention encompasses any other type of source animal with oneor more modifications (genetic or otherwise) that serve(s) to reduceimmunogenicity and/or immunological rejection, singularly or incombination.

In some embodiments, preterm swine fetuses and neonatal piglets arederived as offspring from DPF Closed Colony, α-1,3-galactosyltransferase[Gal-T] knockout pigs, as shown and described herein in accordance withthe present invention.

Such preterm swine fetuses and neonatal piglets are utilized as a sourcefor cells, tissues and organs for xenotransplantation therapies,including, but not limited to, in regenerative or direct transplantationtherapies. It will be understood that such cells, tissues and organs canbe utilized as fresh or following cryopreservation in accordance withthe present invention (e.g., cryopreservation in the range of −80° C.).

In one aspect, mesenchymal cells, pluripotent cells, stem cells and/orother cells that have not differentiated are harvested from such pretermswine fetuses and utilized for regenerative therapies and othertherapies as described herein, whereas such undifferentiated cells canbe found in high proportion in swine fetuses as well as in neonatalpiglets. Since these cells are derived from fetuses earlier along thegestation period, they are less differentiated and more pliable whichoffers greater potential for regenerative therapies. Furthermore, sincethese cells may be derived from DPF Closed Colony,α-1,3-galactosyltransferase [Gal-T] knockout pigs, as shown anddescribed herein, they do not possess aggravating immunogenic,pathogenic and/or other aggravating factors causing rejection by thehuman immune system, and the cells will persist and differentiate insidea human recipient offering regain of function of growth of model tissueusing these genetic and cellular building blocks.

By way of example, such cells may be utilized to generate an array oforgans and/or tissues, through regenerative cell-therapy methods knownin the art (e.g., through utilization of biological scaffolds), forxenotransplantation including, but not limited to, skin, kidneys, liver,brain, adrenal glands, anus, bladder, blood, blood vessels, bones,brain, brain, cartilage, ears, esophagus, eye, glands, gums, hair,heart, hypothalamus, intestines, large intestine, ligaments, lips,lungs, lymph, lymph nodes and lymph vessels, mammary glands, mouth,nails, nose, ovaries, oviducts, pancreas, penis, pharynx, pituitary,pylorus, rectum, salivary glands, seminal vesicles, skeletal muscles,skin, small intestine, smooth muscles, spinal cord, spleen, stomach,suprarenal capsule, teeth, tendons, testes, thymus gland, thyroid gland,tongue, tonsils, trachea, ureters, urethra, uterus, uterus, and vagina,areolar, blood, adenoid, bone, brown adipose, cancellous, cartaginous,cartilage, cavernous, chondroid, chromaffin, connective tissue, dartoic,elastic, epithelial, epithelium, fatty, fibrohyaline, fibrous, Gamgee,Gelatinous, Granulation, gut-associated lymphoid, Haller's vascular,hard hemopoietic, indifferent, interstitial, investing, islet,lymphatic, lymphoid, mesenchymal, mesonephric, mucous connective,multilocular adipose, muscle, myeloid, nasion soft, nephrogenic, nerve,nodal, osseous, osteogenic, osteoid, periapical, reticular, retiform,rubber, skeletal muscle, smooth muscle, and subcutaneous tissue.

Accordingly, preterm swine fetuses and neonatal piglets may be utilizedas a source of tissue, cells and organs in accordance with the presentinvention based on their characteristics as compared to adult swine.

Closed Colonies General Closed Colony

Referring now to FIG. 37, in one aspect, animals are secured from theoutside to consider as candidates to add to the General Closed Colony128 that is housed within the SAF 100 to help propagate the DPF ClosedColony 102 also housed within the SAF 100 in a separate isolation area152. Transportation of the animals secured from the outside to the SAFis controlled to mitigate exposure to potential infectious agents. Suchmitigation techniques include, but are not limited to, using asterilized HEPA filtered cage during transport using a van cleaned withchlorhexidine and containing no other animals.

Candidate animals are initially quarantined to check health status andsuitability for intake into the General Closed Colony 128. For example,in some aspects, animals coming from the outside are first housed in aquarantine intake area 130 within the SAF and accompanied by a completehealth record (including, but not limited to, date of birth,vaccinations, infections, and antibiotic history), pedigree, and resultsof genetic tests. These animals reside in the quarantine intake area 130for at least seven (7) days as the accompanying records are evaluatedand other health screening measures are taken, including screening forsome infectious agents.

In some aspects, animals with poor health, questionable medical status,or are not able to be treated for such medical issues, will not beaccepted into the General Closed Colony 128 and/or will otherwise beculled from the quarantine area 130. Examples of acceptance criteriainclude, but are not limited to: (a) source animals are not born withany congenital defect that was unanticipated from the herd and thatcould have impacted the quality of health of the animal; (b) sourceanimals have received all vaccinations according to age and thevaccinations were killed agents; (c) any infections that occurred in thesource animal's lifetime have been reviewed as well as the clinicalintervention, and it was determined that the infection and any treatment(if applicable) did not impact the quality of the health of the animal;(d) results of the surveillance testing has been reviewed and it hasbeen verified that the source animal has been tested within the last 3months (with all source animals tested at sacrifice and all tests mustbe negative); (e) if the animal was injured in any way which requiredmedical attention, a review has been conducted and it has been confirmedthat the impact of the injury and the medical intervention (ifapplicable) had no impact on the health of the animal; and/or (f) PERVtests have been performed and results recorded.

In some aspects, animals that pass this screening process and timetableare moved out of the quarantine intake area 130 and into a generalholding area 132 within the SAF 100 to join or create an existing ornewly formed General Closed Colony 128. It will be understood that thegeneral holding area 132 is kept under closed colony conditionssubstantially similar to the conditions applied to the DPF Closed Colony102 in the DPF Isolation Area 152.

It will be further understood that, excluding their offspring, candidateanimals secured from the outside will never become members of the DPFClosed Colony. Piglets from the General Closed Colony 128 animals willbe utilized to create and/or propagate the DPF Closed Colony as furtherdescribed herein.

DPF Closed Colony Pregnant Sows and DPF Piglets

In one aspect, pregnant sows 134 (or gilts) are obtained from theoutside or from the General Closed Colony 128 to produce piglets tocreate and/or add to the DPF Closed Colony 102 herd. For example, in oneaspect, sows 134 are placed in a sow quarantine area 136 within the SAFuntil the time to give birth, in this aspect via Cesarean section inorder to avoid exposing the piglet to potential pathogens, includingPorcine Cytomegalovirus (pCMV). Contraction of pCMV in piglets can occurwhen the piglets travel through the vagina of the sow during naturalbirth. The piglets, by virtue of their birthing through Cesarean sectionas described herein, prevents such contraction and the piglets producedthrough the methods described herein are pCMV-free.

Prior to the Cesarean section procedure, for example the morning of theprocedure, an operating room 138 within the SAF 100 prepared accordingto standard operating room protocols in a sterile environment with 2sides: Side A 140 for the Cesarean section of the sow, and Side B 142 toreceive the piglets 144 that are candidates to either found or add tothe DPF Closed Colony.

The sow 134 is brought into the operating room 138 for captive bolteuthanasia. Immediately following this, the sow 134 is placed in theleft lateral decubitus position and the abdomen and torso are preppedwidely with chlorhexidine and draped in a sterile fashion. A flankincision is expeditiously made and the abdominal muscles are split inorder to gain access into the peritoneum. The uterus is exteriorized,incised and the piglets 144 are removed after doubly clamping anddividing the umbilical cord. Immediate execution of the surgicalprocedures following captive bolt euthanasia is critical to the survivalof the piglets 144.

Infection controls for the piglets 144 are implemented at birth. Thepiglets 144 are placed in a warmed 1% chlorhexidine (or othersterilization agent, such as betadine) in sterile saline bath solutionand then passed over to piglet handlers to a resuscitation area 148 forresuscitation, rewarming and gavage feeding of the first dose ofcolostrum. The sow's 134 carcass is closed by staff with suture anddisposed of following appropriate procedures.

The piglets 144 are subsequently quarantined in a separate sterilepiglet quarantine room 150 then transferred to a designated pathogenfree isolation area (“DPF Isolation Area”) 152 to either create or jointhe DPF Closed Colony 102. It will be understood that the DPF IsolationArea 152 can be of any size suitable to manage and maintain the DPFClosed Colony to the extent needed for breeding, rearing, birthing,harvesting, and overall management as described herein.

In one aspect, the DPF Isolation Area 152 that supports the DPF ClosedColony is a restricted access, positive-pressure barrier isolationsuite, approximately 500 ft², with an animal husbandry capacity tosupport at least 9 animals (up to 20 kg each), inside the larger SAF100. It will be understood that the DPF Isolation Area 152 can besignificantly larger than this, and can include multiple areas(including, but not limited to, multiple rooms and suites), depending onthe need of the number of source animals and demand for products, inaccordance with the products and methods as described herein.

In some aspects, tracking of piglets is performed and piglets arehandled under designated pathogen free conditions in the DPF IsolationArea 152. For example, handling of piglets is performed wearing personalprotective equipment (“PPE”) in the DPF Isolation Area 152, includingface mask, gloves, shoe covers, and hair bonnet. The animals are handledby clean personnel, personnel who have not entered any animal room orfacility where other swine are housed. For tracking, piglets are earnotched 3 days after birth and ear tagged with hand-labeled plastic eartags at weaning (usually 3-5 weeks).

It will be understood that some piglets are raised in the DPF ClosedColony 102 in the DPF Isolation Area 152 as a source forxenotransplantation products, and some piglets in the DPF Closed Colony102 are allowed to mature and be used to propagate the General ClosedColony 128. In the event of propagation of the General Closed Colony128, the matured animal is removed from the DPF Isolation Area 152 andadded to the General Closed Colony 128 for breeding. Since the DPFIsolation Area 152 is controlled to be DPF, once these or any otheranimals leave DPF Isolation Area 152, those animals never return to theDPF Isolation Area 152.

Precautions are taken to prevent the exposure of any animals within theDPF Closed Colony 102 to contamination (for example, blood, bloodproducts or tissues obtained from animals outside the DPF Closed Colony102). If any animals within the DPF Closed Colony 102 are inadvertentlyexposed to blood, blood products, or tissues obtained from animalsoutside the DPF Closed Colony 102, those animals are removed from theDPF Closed Colony 102 and will never return to the DPF Closed Colony102. Aseptic techniques and sterile equipment for all parenteralinterventions are used, and routine procedures such as vaccinations,treatment with drugs or biologics, phlebotomy, and biopsies areperformed. The DPF Isolation Area 152 is restricted by card access onlyto specially authorized and trained staff.

In another aspect of the invention, in some aspects, newborn piglets arehandled and hand-reared by trained and gowned staff in the DPF IsolationArea 152 to ensure their health and that they are maintained asdesignated pathogen free.

Propagation

The DPF Closed Colony 102 can be propagated in multiple ways. Forexample, as described herein, sows 134 may be taken from the outside orGeneral Closed Colony 128, quarantined, and have their piglets 144delivered via Cesarean section, with the piglets resuscitated,sterilized, quarantined, and placed into the DPF Isolation Area 152.Newborn piglets may be maintained at 26-30° C. or 80-85° F. In someaspects, heat lamps are used to keep animals warm. Newborn piglets areinitially housed in sterilized medium crates in the SAF with steriletowels/drapes on the bottom.

The DPF Closed Colony 102 may also be propagated in other ways. Forexample, in one aspect, the DPF Closed Colony 102 is propagated throughnatural intercourse amongst the animals in the DPF Closed Colony 102occurring entirely within the DPF Isolation Area 152. It will beunderstood that pregnancies may also occur in the DPF Closed Colony 102within the DPF Isolation Area 152 as a result of artificial inseminationor other breeding techniques that do not involve natural intercourse.

In such aspects, pregnant sows 154 (or gilts) in the DPF Closed Colony102 within the DPF Isolation Area 152 carry the entire pregnancy andpiglets are delivered through live vaginal birth and Caesarian sectionis not necessary. Importantly, the piglets resulting from naturalintercourse and live vaginal birth within the DPF Isolation Area 152 aredesignated pathogen free, including no infection by pCMV.

Following the live vaginal birth, piglets are immediately taken awayfrom the sow to prevent the sows from harming the piglets. The pigletsare then hand-reared from birth by humans within the DPF Isolation Area152 in the methods as described herein.

In the case of mating in the DPF Closed Colony 102 or General ClosedColony 128, the breeding of swine disclosed herein is typicallyhomozygous to homozygous breeding. Females are given hormones two weeksbefore gestation then throughout pregnancy. Furthermore, as with the DPFClosed Colony 102, the General Closed Colony 128 may also be propagatedthrough natural intercourse amongst the animals in the General ClosedColony 128, and may also occur as a result of artificial insemination orother assisted reproductive technologies (ARTs) that do not involvenatural intercourse.

Various techniques have been developed and refined to obtain a largenumber of offspring from genetically superior animals or obtainoffspring from infertile (or subfertile) animals. These techniquesinclude: artificial insemination, cryopreservation (freezing) of gametesor embryos, induction of multiple ovulations, embryo transfer, in vitrofertilization, sex determination of sperm or embryos, nuclear transfer,cloning, etc.

Artificial insemination (AI) has been used to obtain offspring fromgenetically superior males for more than 200 years. Improvements inmethods to cryopreserve (freeze) and store semen have made AI accessibleto more livestock producers. In the same manner as cryopreservation ofsemen, embryo freezing allowed for the global commercialization ofanimals with high genetic qualities.

Multiple ovulation and embryo transfer: Development of embryo transfertechnology allows producers to obtain multiple progeny from geneticallysuperior females. Depending on the species, fertilized embryos can berecovered from females (also called embryo donors) of superior geneticmerit by surgical or nonsurgical techniques. The genetically superiorembryos are then transferred to females (also called embryo recipients)of lesser genetic merit. In cattle and horses, efficient techniquesrecover fertilized embryos without surgery, but only one or sometimestwo embryos are produced during each normal reproductive cycle. In swineand sheep, embryos must be recovered by surgical techniques. To increasethe number of embryos that can be recovered from genetically superiorfemales, the embryo donor is treated with a hormone regimen to inducemultiple ovulations, or superovulation.

In vitro Fertilization: As an alternative to collecting embryos fromdonor animals, methods have been developed recently to produce embryosin vitro (in the laboratory). The methods are also called in vitroembryo production. Immature oocytes (female eggs) can be obtained fromovaries of infertile or aged females, or from regular embryo donors(described above). Ovum (egg) pick up is a nonsurgical technique thatuses ultrasound and a guided needle to aspirate immature oocytes fromthe ovaries. Once the immature oocytes have been removed from the ovary,they are matured, fertilized, and cultured in vitro for up to seven daysuntil they develop to a stage that is suitable for transfer or freezing.

Since the mid 1980s, technology has been developed to transfer thenucleus from either a blastomere (cells from early, and presumablyundifferentiated cleavage stage embryos) or a somatic cell (fibroblast,skin, heart, nerve, or other body cell) to an enucleated oocyte(unfertilized female egg cell with the nucleus removed). This “nucleartransfer” produces multiple copies of animals that are themselves nearlyidentical copies of other animals (transgenic animals, geneticallysuperior animals, or animals that produce high quantities of milk orhave some other desirable trait, etc.). This process is also referred toas cloning. To date, somatic cell nuclear transfer has been used toclone cattle, sheep, pigs, goats, horses, mules, cats, rabbits, rats,and mice.

The technique involves culturing somatic cells from an appropriatetissue (fibroblasts) from the animal to be cloned. Nuclei from thecultured somatic cells are then microinjected into an enucleated oocyteobtained from another individual of the same or a closely relatedspecies. Through a process that is not yet understood, the nucleus fromthe somatic cell is reprogrammed to a pattern of gene expressionsuitable for directing normal development of the embryo. After furtherculture and development in vitro, the embryos are transferred to arecipient female and ultimately result in the birth of live offspring.The success rate for propagating animals by nuclear transfer is oftenless than 10 percent and depends on many factors, including the species,source of the recipient ova, cell type of the donor nuclei, treatment ofdonor cells prior to nuclear transfer, the techniques used for nucleartransfer, etc.

Most commonly used ARTs rely on fertilization as a first step. Thisjoining of egg and sperm is accompanied by the recombination of thegenetic material from the sire and dam, and is often referred to as“shuffling the genetic deck.” It will be understood that these breedingtechniques can be used either within the DPF Closed Colony, as abreeding step within the DPF Isolation Area 152, or could be used as abreeding step for females in the General Closed Colony and/or from theoutside.

In the case of utilization of ART to impregnate females in the GeneralClosed Colony, and/or a female from the outside, the birthing of pigletsfrom such females can be as described herein, i.e., sows 134 may betaken from the outside or General Closed Colony 128, quarantined, andhave their piglets 144 delivered via Cesarean section, with the pigletsresuscitated, sterilized, quarantined, and placed into the DPF IsolationArea 152.

Maintenance of Closed Colonies

Designated pathogens may include any number of pathogens, including, butnot limited to, viruses, bacteria, fungi, protozoa, parasites, and/orprions (and/or other pathogens associated with transmissible spongiformencephalopathies (TSEs)). Designated pathogens could include, but not belimited to, any and all zoonotic viruses and viruses from the followingfamilies: adenoviridae, anelloviridae, astroviridae, calicivirdae,circoviridae, coronaviridae, parvoviridae, picornaviridae, andreoviridae.

Designated pathogens could also include, but not be limited to,adenovirus, arbovirus, arterivirus, bovine viral diarrhea virus,calicivirus, cardiovirus, circovirus 2, circovirus 1, coronavirus,encephalomyocarditus virus, eperytherozoon, Haemophilus suis, herpes andherpes-related viruses, iridovirus, kobuvirus, leptospirillum, listeria,mycobacterium TB, Mycoplasma, orthomyxovirus, papovirus, parainfluenzavirus 3, paramyxovirus, parvovirus, pasavirus-1, pestivirus,picobirnavirus (PBV), picornavirus, porcine circovirus-like(po-circo-like) virus, porcine astrovirus, porcine bacovirus, porcinebocavirus-2, porcine bocavirus-4, porcine enterovirus-9, porcineepidemic diarrhea virus (PEDV), porcine polio virus, porcinelymphotropic herpes virus (PLHV), porcine stool associated circularvirus (PoSCV), posavirus-1, pox virus, rabies-related viruses, reovirus,rhabdovirus, rickettsia, sapelovirus, sapovirus, Staphylococcus hyicus,Staphylococcus intermedius, Staphylococcus epidermidis,coagulase-negative staphylococci, suipoxvirus, swine influenza, teschen,torovirus, torque teno sus virus-2 (TTSuV-2), transmissiblegastroenteritus virus, vesicular stomatitis virus, and/or any and/or allother viruses, bacteria, fungi, protozoa, parasites, and/or prions(and/or other pathogens associated with TSEs). In some aspects,particularly in swine herds, testing for TSEs is not performed becauseTSEs are not reported in natural conditions in swine. In other aspects,testing for TSEs is performed as part of the methods of the presentdisclosure.

There are huge numbers of pathogens that could possibly be tested for inanimal herds, and there is no regulatory guidance or standard, orunderstanding in the field as to what specific group of pathogens shouldbe tested for in donor animals, and which specific group of pathogensshould be removed from donor animal populations in order to ensure safeand effective xenotransplantation. In other words, before the presentdisclosure, there was no finite number of identified, predictablepathogens to be tested for and excluded.

Importantly, the present disclosure provides a specific group ofpathogens identified by the present inventors that are critical toexclude for safe and effective xenotransplantation, as set forth in thefollowing Table 1.

TABLE 1 Test Pathogen Parasite Fecal Float Ascaris speciesCryptosporidium species Echinococcus Strongyloids sterocolis Toxoplasmagondii Brucella BAPA (buffered Brucella suis acidified plateagglutination test) Lepto6 Screen Leptospira species M Hyo MycoplasmaHyopneumoniae PRRS x3 ELISA Porcine Reproductive and RespiratorySyndrome Virus (PRRSV) PRVgb Test Pseudorabies TGE/PRCV Test PorcineRespiratory Coronavirus Toxoplasmosis ELISA Toxoplasma Gondii PorcineCytomegalovirus Porcine CMV PCR Porcine Influenza PCR Porcine InfluenzaA Nasal swab Bordetella bronchiseptica Skin culture Coagulase-positivestaphylococci Skin culture Coagulase-negative staphylococci Skin cultureLivestock-associated methicillin resistant Staphylococcus aureus (LAMRSA) Skin culture Microphyton and Trichophyton spp. Porcine EndogenousPorcine Endogenous Retrovirus (PERV) Retrovirus RT-PCR Assay C (PERV C)

In certain aspects, a product of the present disclosure is sourced fromanimals having antibody titer levels below the level of detection for aplurality of or all of the pathogens discussed in the presentdisclosure. In certain aspects, subjects transplanted with a product ofthe present disclosure are tested and found to have antibody titerlevels below the level of detection for a plurality of or all of thepathogens discussed in the present disclosure.

In some aspects, the present disclosure includes a method of testing fora specific group of pathogens consisting of no more than 18-35, e.g.,35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or18 pathogens, the specific group of pathogens including each of thepathogens identified in Table 1. In some aspects, the present disclosureincludes creating, maintaining and using donor animals that are free ofthe 18-35, e.g., 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22,21, 20, 19, or 18 pathogens, the specific group of pathogens includingeach of the pathogens identified in Table 1.

As described herein, piglets born via live vaginal birth within the DPFClosed Colony 102 are not infected with pCMV, but are nonetheless testedfor pCMV on a continuous basis. Testing for Porcine Cytomegalovirus(pCMV) and Porcine Endogenous Retrovirus (PERV), should be routine andcontinuous for screening and maintenance as described herein, and shouldoccur routinely and continuously for the DPF Closed Colony. In someaspects of the present invention, the source animals described hereinare positive for PERV A and B only, and some are positive for PERV A, B,and C. In other aspects, the source animals are free of PERV A, B and/orC (through utilization of CRISPR and other techniques).

With respect to PERV, it is understood that most, if not all, swine areknown to be positive for PERV A and B. While PERV is recognized, therisk of transmission of PERV from treatment with swine derived tissue isexpected to be rare. To date eight PERV mRNAs are expressed in allporcine tissues and in all breeds of swine and preclinical and clinicalxenotransplantation studies of humans exposed to pig cells, tissues, andorgans including pancreatic islets have failed to demonstratetransmission of PERV. See, e.g., Morozov V A, Wynyard S, Matsumoto S,Abalovich A, Denner J, Elliott R, “No PERV transmission during aclinical trial of pig islet cell transplantation,” Virus Res 2017;227:34-40. In the unlikely event that a human infection should occur,PERV is susceptible in vitro to nucleoside and non-nucleoside reversetranscriptase inhibitors in common clinical use. See, e.g., Wilhelm M,Fishman J A, Pontikis R, Aubertin A M, Wilhelm F X, “Susceptibility ofrecombinant porcine endogenous retrovirus reverse transcriptase tonucleoside and non-nucleoside inhibitors,” Cellular & Molecular LifeSciences 2002; 59:2184-90; Schuurman, H., “Regulatory aspects ofclinical xenotransplantation,” Int. J. Surg., 23, (2015), pp. 312-321.Experimental data using the xenotransplantation product of the presentdisclosure indicated that PERV genetic material was not detected in therecipient's organs and that porcine DNA and cells did not migrate intothe circulation of the recipient from the xenotransplanted organ.

The DPF Closed Colony 102 is maintained to ensure that the animalsremain designated pathogen free and that appropriate standards of animalcare and well-being are applied at all levels of the SAF 100 (i.e.,breeding, maintenance, propagation). No animal is permitted into the DPFClosed Colony if it or a parent has tested positive for any of thepathogens in Table 1. For example, continuous testing for pathogens andother biological markers occurs including the numerous pathogensidentified herein (including, but not limited to, pCMV and otherpathogens). Environmental and blood samples are collected as necessaryfor genotyping and testing for pathogens. Test result(s) obtained forpathogens or other health concerns are evaluated by the facilityveterinarian who may recommend follow-up testing and observations, andquarantine of the facility or areas (e.g., rooms, suites or other areas)within a facility as needed. Careful documentation of any antimicrobialagents used during routine care of the source animals should bemaintained, and exclusive use of killed vaccines used. Examples ofantimicrobial agents include cefazolin, bacitracin, neomycin, andpolymyxin.

In some aspects, routine health surveillance and screening for pathogens(e.g., adventitious agents) of source animals is performed every 3months. Samples of serum, nasal swabs, and stool for each animal in theGeneral and DPF Closed Colonies are obtained and provided for analyticaltests for detection of such pathogens every 3 months. Source animalsamples of serum, nasal swabs, and stool for testing are obtainedimmediately after euthanasia via captive bolt and evaluated as disclosedherein including one or more of: conducting a sterility assay andconfirming that aerobic and anaerobic bacteria do not grow in thesterility assay; conducting a Mycoplasma assay and confirming thatMycoplasma colonies do not grow in the Mycoplasma assay; conducting anendotoxin assay and confirming that the biological product is free ofendotoxins in the endotoxin assay, conducting the MTT-reduction assayand confirming that the product has at least 50% cell viability in theMTT-reduction assay; conducting flow cytometry and confirming that theproduct does not have galactosyl-a-1,3-galactose epitopes as determinedby the flow cytometry; conducting pathogen-detection assays specific for18 to 35 pathogens and confirming that the product is free of Ascarisspecies, Cryptosporidium species, Echinococcus, Strongyloids sterocolis,Toxoplasma gondii, Brucella suis, Leptospira species, Mycoplasmahyopneumoniae, porcine reproductive and respiratory syndrome,pseudorabies, Staphylococcus species, Microphyton species, Trichophytonspecies, porcine influenza, porcine cytomegalovirus, arterivirus,coronavirus, Bordetella bronchiseptica, and Livestock-associatedmethicillin-resistant Staphylococcus aureus.

In some aspects, all swine undergo routine health monitoring, whichincludes documentation of all illnesses, medical care, procedures, drugsadministered, vaccinations, physical examinations, any treatmentsreceived, and general health assessments and observations each day attime of feeding with a visual health inspection indicating the animal isable to stand, move freely and appears clinically normal, as well asobservations relating to the animal's appearance, activity and appetite,recording on the Animal Husbandry Log any deficiencies. In some aspects,animals are vaccinated against Mycoplasma hyopneumoniae, HemophilusParasuis, Streptococcus Suis, Pasteurella Multocida, Bordatellabronchiseptica and Erysipelothrix rhusiopathiae. All swine six months orolder may be vaccinated against Erysipelothrix rhusiopathiae, Leptospira(Canicola-Grippotyphosa-Hardjo-Icterohaemorrhagiae-Pomona), Influenzaand Parvovirus. Repeat vaccination may be performed, e.g., every sixmonths.

In some aspects, health monitoring will normally be performed as part ofdaily husbandry procedures for cleaning and feeding to minimize entryinto swine holding areas (e.g., rooms, suites or other areas). Prior toentering, personnel must wear personal protective equipment (PPE) andensure that their footwear is free from gross contamination (e.g.visible dirt or mud). They will then don disposable shoe/boot coversprior to entry. Personnel in contact with any animals not housed in thedesignated pathogen free facility will change PPE if contaminated. Allimplements (shovel, other necessary tools) will undergo chlorhexidineimmersion of no less than 2 minutes if exogenous to vivarium and judgednecessary. Solid waste and soiled bedding is removed. Animal holdingareas are sanitized with diluted Quat-PV or bleach a minimum of onceevery two weeks.

In some aspects, bedding is replaced daily using irradiated bedding woodshavings. The replacement amount is an approximate equal amount to thatwhich was removed. All bedding is completely replaced on a weekly basisat a minimum. Daily activities including health status checks, cleaningand water levels are documented in the Animal Husbandry log.Appropriately labeled trash and biological waste is picked up by staffdaily and incinerated.

With regard to piglet, newborns are handled and cared for by trained andgowned staff in an isolation suite. All supplies, room and crates aresanitized prior to housing of the piglets. Sterile drapes and towels areused to line the bottom of the crates. Room temperature is controlled to80-85° F. Animals crates are maintained at 85-95° F. through the use ofheat lamps. Piglets are maintained in the crates through the first 2weeks after which time piglets are housed on the floor with irradiatedwood shavings. Crates are cleaned daily and shavings are removed andreplenished daily. Piglets are initially fed fresh-made, sterilecolostrum (Bovine Colostrum IgG formulated for swine, Sterling NursemateASAP or equivalent) using a feeding tube every 1 to 2 hours until pigletis self-feeding from feeder. During the early days, the piglet isweighed twice a day and well-being is checked and recorded twice a day.Starting at day 14, piglets are fed 3 times per day with a Milk Replacer(Ralco Birthright or equivalent) that is further supplemented withirradiated piglet grain (antibiotic free creep feed, Blue Seal 813 orequivalent). The amount each piglet eats at each feeding is recorded.Vaccinations, genotyping, ear notching, and needle teeth trimming areperformed within the first 7 days after birth of the piglet. In someaspects, vaccines use killed agents. Piglets are vaccinated againstMycoplasma hyopneumoniae, Hemophilus parasuis, Streptococcus suis,Pasteurella multocida, Bordatella bronchiseptica and Erysipelothrixrhusiopathiae at day 7 after birth, with a booster vaccination at 28days of age. In one aspect, vaccines are killed agents. All swine sixmonths or older are vaccinated against Erysipelothrix rhusiopathiae,Leptospira (Canicola-Grippotyphosa-Hardjo-Icterohaemorrhagiae-Pomona),Influenza and Parvovirus. Repeat vaccination is performed every sixmonths.

The source animals for the xenotransplantation product are maintained ina positive pressure, biocontainment establishment, under specificisolation-barrier conditions governed by standard operation proceduresadopted by the managers of the given program, and receive specializedcare, under controlled conditions in order to mitigate adventitiousagents. To ensure the welfare of the closed colony of source animalsintended for xenotransplantation use, the SAF, personnel, and thecaretakers of source animals adhere to procedures for animal husbandry,tissue harvesting, and sacrifice of animals. The source animals arehoused in a positive pressure, biocontainment establishment, underspecific isolation-barrier conditions.

In some aspects, food and bedding are delivered to a loading dock,transported, and stored in a specific feed room off of the clean cagewash area accessible only to staff in the inner hallway. All bedding andfeed are sterilized by irradiation and double bagged to insuresterility. Feed used for the piglets and more mature animals is definedgrain feed by a specific manufacturer. It does not contain any cattleprotein. Water supply is provided either by use of the facility sterilesystem or purchased sterile water which is dispensed into sterile pans.Records for storage and delivery of feed, water, and other consumablesare maintained, and include manufacturer, batch numbers, and otherpertinent information, per protocol.

In some aspects, animal records are maintained to describe the feedprovided to source animals for at least two generations before their useas a source for live tissues, organs and/or cells used inxenotransplantation. This includes source, vendor, and the type of feedused (including its contents). Use of feed that has been derived fromanimals is prohibited. Source animals are not provided feeds containinganimal proteins or other cattle materials that are prohibited by the FDAfeed ban as expanded in 2008 as source animals (21 CFR 589.2000) orfeeds containing significant drug contamination or pesticide orherbicide residues for source animals (21 CFR 589.2001).

In some aspect, purified water is provided in sufficient quality toprevent unnecessary exposure of animals to infectious pathogens, drugs,pesticides, herbicides, and fertilizers. Newborn animals are providedcolostrum specifically qualified for herd qualification. In someaspects, Bovine Colostrum IgG formulated for swine, Sterling NursemateASAP or equivalent is used to feed newborn animals.

Biological Products Derived from DPF Closed Colony Biological Products

As described herein, biological products for xenotransplantation arederived from source animals produced and maintained in accordance withthe present invention, including from the DPF Closed Colony 102 asdescribed herein. Such biological products include, but are not limitedto, liver, kidney, skin, lung, heart, pancreas, intestine, nerve andother organs, cells and/or tissues.

The present disclosure provides a continuous manufacturing process for axenotransplantation product that has reduced immunogenicity, reducedantigenicity, increased viability, increased mitochondrial activity, aspecifically required pathogen profile, and unexpectedly long shelf-lifein xenotransplantation tissues subject to cryopreservation. Thecontinuous manufacturing process is surprisingly and unexpectedlyeffective in avoiding hyperacute rejection, delayed xenograft rejection,acute cellular rejection, chronic rejection, cross-species transmissionof diseases, cross-species transmission of parasites, cross-speciestransmission of bacteria, cross-species transmission of fungi, andcross-species transmission of viruses. The continuous manufacturingprocess is surprisingly and unexpectedly effective in creating a closedherd in which the donor animals survive normally without detectablepathological changes.

Harvesting of such biological products occurs in a single, continuous,and self-contained, segregated manufacturing event that begins with thesacrifice of the source animal through completion of the production ofthe final product. The animal is euthanized via captive bolt euthanasia,may be moved, if necessary, in a sterile, non-porous bag, to anoperating room where the procedure to harvest biological product fromthe source animal will occur. All members of the operating team shouldbe in full sterile surgical gear, e.g., dressed in sterile dress tomaintain designated pathogen free conditions prior to receiving thesource animal and in some instanced be double-gloved to minimizecontamination, and surgical areas and tools are sterilized. The sourceanimal is removed from the bag and container in an aseptic fashion. Thesource animal is scrubbed by operating staff, e.g., for at least 1-10minutes with antiseptic, e.g., Chlorhexidine, brushes over the entirearea of the animal where the operation will occur, periodically pouringChlorhexidine over the area to ensure coverage. Surgical area(s) of theanimal are scrubbed with opened Betadine brushes and sterile water rinseover the entire area of the animal where the operation will occur for,e.g., 1-10 minutes. For surgery, operators will be dressed in steriledress in accordance with program and other standards to maintaindesignated pathogen free conditions. All organs, cells or tissue fromthe source animal that will be used for xenotransplantation is harvestedwithin 15 hours of the animal being sacrificed.

Biological products can also include, but are not limited to, thosedisclosed herein (e.g., in the specific examples), as well as any andall other tissues, organs, and/or purified or substantially pure cellsand cell lines harvested from the source animals. In some aspects,tissues that are utilized for xenotransplantation as described hereininclude, but are not limited to, areolar, blood, adenoid, bone, brownadipose, cancellous, cartaginous, cartilage, cavernous, chondroid,chromaffin, connective tissue, dartoic, elastic, epithelial, Epithelium,fatty, fibrohyaline, fibrous, Gamgee, Gelatinous, Granulation,gut-associated lymphoid, Haller's vascular, hard hemopoietic,indifferent, interstitial, investing, islet, lymphatic, lymphoid,mesenchymal, mesonephric, mucous connective, multilocular adipose,muscle, myeloid, nasion soft, nephrogenic, nerve, nodal, osseous,osteogenic, osteoid, periapical, reticular, retiform, rubber, skeletalmuscle, smooth muscle, and subcutaneous tissue. In some aspects, organsthat are utilized for xenotransplantation as described herein include,but are not limited to, skin, kidneys, liver, brain, adrenal glands,anus, bladder, blood, blood vessels, bones, cartilage, cornea, ears,esophagus, eye, glands, gums, hair, heart, hypothalamus, intestines,large intestine, ligaments, lips, lungs, lymph, lymph nodes and lymphvessels, mammary glands, mouth, nails, nose, ovaries, oviducts,pancreas, penis, pharynx, pituitary, pylorus, rectum, salivary glands,seminal vesicles, skeletal muscles, skin, small intestine, smoothmuscles, spinal cord, spleen, stomach, suprarenal capsule, teeth,tendons, testes, thymus gland, thyroid gland, tongue, tonsils, trachea,ureters, urethra, uterus, and vagina.

In some aspects, purified or substantially pure cells and cell linesthat are utilized for xenotransplantation as describe herein include,but are not limited to, blood cells, blood precursor cells, cardiacmuscle cells, chondrocytes, cumulus cells, endothelial cells, epidermalcells, epithelial cells, fibroblast cells, granulosa cells,hematopoietic cells, Islets of Langerhans cells, keratinocytes,lymphocytes (B and T), macrophages, melanocytes, monocytes, mononuclearcells, neural cells, other muscle cells, pancreatic alpha-1 cells,pancreatic alpha-2 cells, pancreatic beta cells, pancreatic insulinsecreting cells, adipocytes, epithelial cells, aortic endothelial cells,aortic smooth muscle cells, astrocytes, basophils, bone cells, boneprecursor cells, cardiac myocytes, chondrocytes, eosinophils,erythrocytes, fibroblasts, glial cells, hepatocytes, keratinocytes,Kupffer cells, liver stellate cells, lymphocytes, microvascularendothelial cells, monocytes, neuronal stem cells, neurons, neutrophils,pancreatic islet cells, parathyroid cells, parotid cells, platelets,primordial stem cells., Schwann cells, smooth muscle cells, thyroidcells, tumor cells, umbilical vein endothelial cells, adrenal cells,antigen presenting cells, B cells, bladder cells, cervical cells, conecells, egg cells, epithelial cells, germ cells, hair cells, heart cells,kidney cells, leydig cells, lutein cells, macrophages, memory cells,muscle cells, ovarian cells, pacemaker cells, peritubular cells,pituitary cells, plasma cells, prostate cells, red blood cells, retinalcells, rod cells, Sertoli cells, somatic cells, sperm cells, spleencells, T cells, testicular cells, uterine cells, vaginal epithelialcells, white blood cells, ciliated cells, columnar epithelial cells,dopaminergic cells, dopaminergic cells, embryonic stem cells,endometrial cells, fibroblasts fetal fibroblasts, follicle cells, gobletcells, keratinized epithelial cells, lung cells, mammary cells, mucouscells, non-keratinized epithelial cells, osteoblasts, osteoclasts,osteocytes, and squamous epithelial cells.

An organ is a group of related cells that combine together to performone or more specific functions within the body. Biologically, skin isthe body's largest and fastest-growing organ, and is classified as theprimary component of the integumentary system, one of the tenmacro-organ systems found in “advanced” animals. Skin fulfills severalcritical roles including regulating temperature, providing a dynamicbarrier to the external world, and serving as a conduit to support animmense network of sensory receptors. The skin performs severalfunctions that are vital to the survival and health of the body. Theskin heals to prevent the loss of blood after wounds, regulates bodytemperature by dissipating heat and as a layer against cold, absorption,secretion, thermal-regulation, sensory detection and orientation, andbarrier protection. In fact, not only has success in transplantation ofskin been recognized to correlate to transplantation of other organs,but skin transplants appear to be more sensitive to rejection than otherorgans, e.g., immune privileged organs such as liver, and skintransplants have even been suggested for use as “sentinel transplants,”i.e., use of skin grafts in a human recipient as early predictors ofrejection of transplanted solid organs in the same recipient. Forexample, as reported in Ali et al. Transplant Proc. 2016 October;48(8):2565-2570, evidence provided by experience with abdominal walltransplantation in some intestinal and multivisceral transplantrecipients suggest that rejection may manifest in the skin componentbefore emergence in the intestinal allograft, providing a “lead time”during which treatment of rejection of the abdominal wall could preventthe emergence of intestinal rejection.

Further, United States Code Title 42, Section 274 and Section 301,explicitly list skin in its formal definition of human organs, i.e.,“‘Human organ,’ as covered by section 301 of the National OrganTransplant Act, as amended, means the human (including fetal) kidney,liver, heart, lung, pancreas, bone marrow and other hematopoieticstem/progenitor cells without regard to the method of their collection,cornea, eye, bone skin, and intestine, including the esophagus, stomach,small and/or large intestine, or any portion of the gastrointestinaltract.” Similarly, the Human Organ Transplant Ordinance (HOTO), aninternationally ratified ordinance to prevent organ trading and protectdonor and recipient rights to self-determination. This globallegislation lists skin—and whole segments of the integumentarysystem—formally as an organ, and more broadly defines an organ as “anypart of the human body consisting of a structured arrangement of tissueswhich, if wholly removed, cannot be regenerated by the body . . . .”Following, the formal medical definition of a transplant is: “theremoval of tissue from one part of the body or from one individual andits implantation or insertion in another especially by surgery.” TheHOTO defines a transplant as “the transfer of an organ from one personto another during a transplant operation, regardless of permanence.”

With regard to skin, grafts typically consist of decellularized and/orreconstituted sheets of homogenized dermis that are used to achievetemporary, superficial wound coverage. Such grafts do not retain theoriginal tissue structure nor the metabolically active, otherwisenaturally present cells, and thus do not become vascularized; nocapillary ingrowth or vessel-to-vessel connections are made.Consequently, immune rejection is not a concern—the skin graft becomes“ejected” rather than rejected by the growth of a complete hostepithelium underneath the graft. Thus, while the term graft can becorrectly applied to such solutions, the primary qualities thatdifferentiate a transplant from a graft are that of heightenedcomplexity, organization, and inclusion of one or more types of tissue.In the present case, a skin transplant is fundamentally differentiatedfrom grafts known in the prior art. For example, a skin xenotransplantis comprised of live cells that perform the same function as thepatient's original skin before eventually experiencing immune-mediatedrejected. Thus, in this context, a skin xenotransplant according to thepresent disclosure is an organ transplant rather than a graft.

In terms of harvesting a biological product from the swine, wherein theharvesting comprises euthanizing the swine and aseptically removing thebiological product from the swine; processing said biological productcomprising sterilization after harvesting using a sterilization processthat does not reduce cell viability to less than 50% cell viability in a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)-reduction assay and does not reduce mitochondrial activity to lessthan 50% mitochondrial activity; and storing the biological product in asterile container; and the non-human animal is a non-transgenicgenetically reprogrammed swine for xenotransplantation of cells, tissue,and/or an organ isolated from the non-transgenic geneticallyreprogrammed swine, the non-transgenic genetically reprogrammed swinecomprising a nuclear genome that has been reprogrammed to replace aplurality of nucleotides in a plurality of exon regions of a majorhistocompatibility complex of a wild-type swine with nucleotides fromorthologous exon regions of a known human major histocompatibilitycomplex sequence from a human capture sequence, wherein saidreprogramming does not introduce any frameshifts or frame disruptions.Further specific aspects, details and examples are provided in thefollowing disclosures and claims and any and all combinations of thoseaspects, details and examples constitute aspects of the presentdisclosure.

In other aspect, Xenogeneic kidneys are derived from a geneticallyengineered, reprogrammed and designated pathogen free swine is producedin accordance with the present invention and transplanted into anon-human primate and a human. It is expected that survival of at leastfourteen months is observed in each of the non-human primate and thehuman. In some aspects, it is expected that survival of at least 24months is observed in each of the non-human primate and the human. Insome aspects, it is expected that survival of at least 36 months isobserved in each of the non-human primate and the human. In someaspects, it is expected that survival of at least 48 months is observedin each of the non-human primate and the human. In some aspects, it isexpected that survival of at least 60 months is observed in each of thenon-human primate and the human.

In another aspect, Xenogeneic lungs are derived from a geneticallyengineered, reprogrammed and designated pathogen free swine produced inaccordance with the present invention and transplanted into a non-humanprimate and a human. It is expected that survival of at least 30 days isobserved in each of the non-human primate and the human. In someaspects, it is expected that survival of at least 3 months is observedin each of the non-human primate and the human. In some aspects, it isexpected that survival of at least 6 months is observed in each of thenon-human primate and the human. In some aspects, it is expected thatsurvival of at least 12 months is observed in each of the non-humanprimate and the human. In some aspects, it is expected that survival ofat least 24 months is observed in each of the non-human primate and thehuman. In some aspects, it is expected that survival of at least 36months is observed in each of the non-human primate and the human. Insome aspects, it is expected that survival of at least 48 months isobserved in each of the non-human primate and the human. In someaspects, it is expected that survival of at least 60 months is observedin each of the non-human primate and the human.

In another aspect, Xenogeneic hearts are derived from a geneticallyengineered, reprogrammed and designated pathogen free swine produced inaccordance with the present invention and transplanted into a non-humanprimate and a human. It is expected that survival of at least 20 monthsis observed in each of the non-human primate and the human. In someaspects, it is expected that survival of at least 24 months is observedin each of the non-human primate and the human. In some aspects, it isexpected that survival of at least 36 months is observed in each of thenon-human primate and the human. In some aspects, it is expected thatsurvival of at least 48 months is observed in each of the non-humanprimate and the human. In some aspects, it is expected that survival ofat least 60 months is observed in each of the non-human primate and thehuman.

In another aspect, Xenogeneic nerve tissues are derived from agenetically engineered, reprogrammed and designated pathogen free swineproduced in accordance with the present invention and transplanted intoa non-human primate and a human. It is expected that survival of atleast 75 days is observed in each of the non-human primate and thehuman. In some aspects, it is expected that survival of at least 3months is observed in each of the non-human primate and the human. Insome aspects, it is expected that survival of at least 6 months isobserved in each of the non-human primate and the human. In someaspects, it is expected that survival of at least 12 months is observedin each of the non-human primate and the human. In some aspects, it isexpected that survival of at least 24 months is observed in each of thenon-human primate and the human. In some aspects, it is expected thatsurvival of at least 36 months is observed in each of the non-humanprimate and the human. In some aspects, it is expected that survival ofat least 48 months is observed in each of the non-human primate and thehuman. In some aspects, it is expected that survival of at least 60months is observed in each of the non-human primate and the human.

In another aspect, Xenogeneic livers are derived from a geneticallyengineered, reprogrammed and designated pathogen free swine produced inaccordance with the present invention and transplanted into a non-humanprimate and a human. It is expected that survival of at least 60 days isobserved in each of the non-human primate and the human. In someaspects, it is expected that survival of at least 3 months is observedin each of the non-human primate and the human. In some aspects, it isexpected that survival of at least 6 months is observed in each of thenon-human primate and the human. In some aspects, it is expected thatsurvival of at least 12 months is observed in each of the non-humanprimate and the human. In some aspects, it is expected that survival ofat least 24 months is observed in each of the non-human primate and thehuman. In some aspects, it is expected that survival of at least 36months is observed in each of the non-human primate and the human. Insome aspects, it is expected that survival of at least 48 months isobserved in each of the non-human primate and the human. In someaspects, it is expected that survival of at least 60 months is observedin each of the non-human primate and the human.

In some embodiments, use of pig livers produced in accordance with thepresent invention to serve as extracorporeal filters for humans aredisclosed. In a study by Levy, et al., “Liver allotransplantation afterextracorporeal hepatic support with transgenic (hCD55/hCD59) porcinelivers: Clinical results and lack of pig-to-human transmission of theporcine endogenous retrovirus,” Transplantation, 69(2):272-280 (2000)(“Levy”), the entire contents of which are incorporated herein byreference, whole organ extracorporeal perfusion of a geneticallymodified transgenic porcine liver was proposed to sustain patientsawaiting human liver transplantation for fulminant hepatic failure. Thepig livers used were reported to be transgenic for human CD55(decay-accelerating factor) and human CD59, however, the livers failedto suppress marked increase of [alpha]-gal antibodies.

In accordance with the present invention, in one aspect, a liver derivedfrom a genetically reprogrammed source animal in accordance with thepresent invention is utilized for extracorporeal perfusion as atemporary filter for a human patient until a patient receives a humantransplant. It will be understood that pigs with additional geneticmodifications may also be utilized, including pigs geneticallyreprogrammed for any number of traits disclosed elsewhere herein.

In one aspect, as shown in FIG. 38, an extracorporeal circuit utilizesan oxygenator (e.g., Minimax Plus® hollow fiber oxygenator), a pump(e.g., Bio-Medicus model 540 Bio-Console® with a BP50 Pediatric BioPump® centrifugal pump), and a warmer (Bio-Medicus model 370 BioCal™Temperature Controller). The circuit also utilizes a roller pump (e.g.,Sarns model 7000; Sarns, Ann Arbor, Mich.) to supplement for lack ofgravity return to the patient. Bridges and clamps are utilized toisolate both the perfused liver and the patient.

In an operating area within the DPF Isolation Area, a source animal isplaced under a general anesthetic (ketamine, xylazine, enflurane) oreuthanized by captive bolt. A hepatectomy is then performed on thesource animal in designated pathogen free conditions.

The livers can be preserved in any number of ways known in the art priorto use as an extracorporeal filter, including, but not limited to, asdisclosed in Levy (e.g., “a 4° C. lactated Ringer's/albumin solution andcannulated in the portal vein (28F Research Medical, model SPC-641-28)and the inferior vena cava (36F Research Medical, model SPC-641-36)”).

The common bile duct can be intubated in any number of ways, including,but not limited to, as set forth in Levy (e.g., “with an intravenousextension tube (Extension Set 30, Abbott Hospitals, Inc., Chicago, Ill.)to allow subsequent quantification of bile production.”)

The liver product derived from the source animal can be packaged andtransported to the location of the procedure in accordance with currentpractice with human donor livers.

The procedure to utilize the liver filtration product can be performed,for example, by percutaneously cannulating a patient's internal jugularvein for venous return with a 12F pediatric arterial cannula (e.g.,Medtronic DLP, Grand Rapids, Mich.) and percutaneously cannulating apatient's femoral vein for venous outflow with a 19F femoral arterycannula (e.g., Medtronic Bio-Medicus, Eden Prairie, Minn.). Thesecannulas are connected to a bypass circuit, having a centrifugal pump(e.g., Bio-Medicus), a heat exchanger (Medtronic Bio-Medicus), anoxygenator (e.g., Medtronic Cardiopulmonary, Anaheim, Calif.), and aroller pump (e.g., Sams) incorporated therein.

This circuit is primed with crystalloids and run for a period of time(e.g., 20 minutes) before the liver obtained from the geneticallyreprogrammed source animal is incorporated at a stabilized flow rate of800 ml/min, maintained in a crystalloid bath occasionally supplementedwith warm solution.

In other aspect, Xenogeneic pancreases are derived from a geneticallyengineered, reprogrammed and designated pathogen free swine is producedin accordance with the present invention Xenogeneic pancreas derivedfrom a genetically reprogrammed swine produced in accordance with thepresent invention is transplanted into a non-human primate and a human.It is expected that survival of at least 20 months is observed in eachof the non-human primate and the human. In some aspects, it is expectedthat survival of at least 24 months is observed in each of the non-humanprimate and the human. In some aspects, it is expected that survival ofat least 36 months is observed in each of the non-human primate and thehuman. In some aspects, it is expected that survival of at least 48months is observed in each of the non-human primate and the human. Insome aspects, it is expected that survival of at least 60 months isobserved in each of the non-human primate and the human.

While the subject matter of this disclosure has been described and shownin considerable detail with reference to certain illustrative aspects,including various combinations and sub-combinations of features, thoseskilled in the art will readily appreciate other aspects and variationsand modifications thereof as encompassed within the scope of the presentdisclosure. Moreover, the descriptions of such aspects, combinations,and sub-combinations is not intended to convey that the claimed subjectmatter requires features or combinations of features other than thoseexpressly recited in the claims. Accordingly, the scope of thisdisclosure is intended to include all modifications and variationsencompassed within the spirit and scope of the following appendedclaims.

In other aspect, Xenogeneic dermal combination product derived from agenetically engineered, reprogrammed and designated pathogen free swineis produced in accordance with the present invention.

Some skin transplantation products for the treatment of burns and otherailments utilize cultured epidermal autografts (see, e.g., productsproduced by Vericel Corporation under the Epicel® brand name). Suchepidermal autografts can be utilized for patients with burns (includingsevere burns) and result in reduced or no rejection in the transplantedepidermal material since the material is derived from the patient's ownskin.

However, such products are limited to the epidermis only, and do notinclude the dermis portion of the skin. Referring to FIG. 39, it will beunderstood that the dermis (which typically accounts for 95% of thethickness of the skin) performs significantly different functions thanthe epidermis (which is the outer portion of the skin that typicallyaccounts for 5% of the thickness of the skin).

Since epidermal autografts alone lack the ability to perform thecritical functions of the dermis, such products are used in combinationwith a viable dermis. In some injuries, the wound bed includes remainingportions of the patient's own dermis, which is the ideal dermis toutilize in a procedure grafting cultured epidermal autografts onto apatient. However, in some cases the burn is more severe, and thepatient's own dermis no longer exists or is no longer viable. In thoseinstances, a different dermis is required since an epidermal autograftalone will not suffice.

In one aspect, a full thickness skin graft wound dressing consisting ofdermal tissue derived from designated pathogen freeα-1,3-galactosyltransferase [Gal-T] knockout swine in accordance withthe present invention is used in conjunction or combination withcultured epidermal autografts. One treatment process utilizing thiscombination is as follows.

A patient with severe burn wounds is taken to an operating room within48-72 hours of injury. A biopsy is taken as soon as possible after thepatient undergoes care, and the epidermis skin cells are isolated andgrown separately according to the known procedures for creating culturedepidermal autografts (see, e.g., products produced by VericelCorporation under the Epicel® brand name).

Depending on how much of the patient's body is damaged, epidermalautografts are taken from healthy areas to treat burned areas and/or tolater create an epidermal autograft mesh used in the grafting process.

Areas of severe burns are treated with the skin products describedherein, e.g., skin products derived from a designated pathogen freeα-1,3-galactosyltransferase [Gal-T] knockout swine produced inaccordance with the present invention. Such treatments comprisetemporary wound coverage until sufficient autografts are utilized totreat the patient long-term.

Prior to application of the epidermal autografts, significantdebridement of wound bed is required to ensure an adequate substrate. Toconfirm a wound bed is ready for an epidermal autograft, apply the skinproducts described herein, e.g., skin products derived from a designatedpathogen free α-1,3-galactosyltransferase [Gal-T] knockout swineproduced in accordance with the present invention to confirm adherence.Once adherence is confirmed, the temporary wound coverage product isremoved, and in some aspects, the wound bed is covered with a meshedautograft, and one or more cultured epidermal autograft products areplaced on top to close the gaps in the autograft mesh.

The debridement may include mechanical debridement, chemicaldebridement, enzymatic debridement, or a combination thereof. Mechanicaldebridement may include surgical excision, e.g., tangential excision toremove thin layers of dermis until healthy tissue is visualized, orfascial excision to remove the full thickness of dermis down to theunderlying fascia. Tangential excision allows less viable tissue to beremoved with the necrotic tissue, but typically results in higher bloodloss, is a larger physiologic stressor than fascial excision, and ismore likely to result in “incomplete” debridement, with some devitalizedtissue remaining in place. In fascial excision, blood loss and operativetime are minimized, but often a large amount of healthy tissue isremoved with the burned tissue. Debriding agents may include agentscapable of cleaning a burn wound by removing foreign material and deadtissue. Many such agents are known. In enzymatic debridement,collagenases or other proteolytic enzymes are employed that break downproteins of the extracellular matrix, allowing devitalized tissue to bewiped away without the need for surgery while preferably leaving healthytissue substantially intact. Enzymatic debridement involves theapplication of proteolytic and optionally other exogenous enzymes to awound surface to break down necrotic tissue. Enzymatic debridement maybe a relatively slow process, carried out over a period of a number ofweeks in combination with other topical preparations, soakings andrepeated dressings. Alternately, rapid enzymatic debridement can beaccomplished using multi-enzyme products, for example, those extractedfrom the stem of the pineapple plant, as disclosed for example in WO98/053850 and WO 2006/0006167, and as provided in the product marketedunder the trade name Debrase®. A procedure for enzymatic debridementgenerally utilizes an enzyme such as bromelain derivatives, debridase,collagenase, papain derivatives, streptokinase, sutilains, fibrinolysin,deoxyribonuclease, krill derivatives, trypsin or combinations thereof.Autolytic debridement relies on enhancing the natural process ofselective liquefaction, separation and digestion of necrotic tissue andeschar from healthy tissue that occurs in wounds due to macrophage andendogenous proteolytic activity. This is achieved by the use ofocclusive, semi-occlusive or moist interactive dressings. Enzymaticdebridement agents include a bromelain enriched enzyme product, othercollagenases, or other enzyme products capable of clearing devitalizedtissue or wound debris. NexoBrid™ (MediWound Ltd.) is one such bromelainenriched product that specifically targets heat-denatured collagen fordegradation, resulting in partial-thickness and full-thickness woundsrequiring a wound coverage or dressing product. Such products andmethods are described in U.S. Pat. Nos. 8,540,983; 8,119,124; 7,128,719;7,794,709; 8,624,077; and US2009/0010910A1, each of which isincorporated by reference herein.

In some aspects, the wound bed may include or be a chronic wound or anacute wound. Chronic wounds include but are not limited to venous legulcers, pressure ulcers, and diabetic foot ulcers. Acute wounds includebut are not limited to burns, traumatic injuries, amputation wounds,skin graft donor sites, bite wounds, frostbite wounds, dermabrasions,and surgical wounds.

In the cases where there is no dermis, skin products derived from adesignated pathogen free α-1,3-galactosyltransferase [Gal-T] knockoutswine produced in accordance with the present invention are utilized.The epidermis is removed from such products (e.g., before dermisharvesting on the pig with a VERSAJET™ Hydrosurgery system), so thatjust the dermis remains. Then, the subject swine dermis is placed on thepatient's subcutaneous tissue, serving as a substrate for the culturedepidermal autograft process described above.

Product Characteristics, Testing and Therapeutic Uses

In some aspects, the xenotransplantation products described anddisclosed herein are temporary, i.e., their use in patients forxenotransplantation is non-permanent, utilized primarily for thetreatment of acute ailments and injuries, able to be utilized for longerperiods of time as compared to products that are not produced inaccordance with the present invention. It will be understood that someof the aspects of the products described and disclosed herein may alsobe permanent or more permanent, with transplanted organs, tissues and/orcells being accepted by human recipients over much longer periods oftime without adverse rejection.

In other aspects, the xenotransplantation products described anddisclosed herein are viable, live cell (e.g., vital, biologicallyactive) products; distinct from synthetic or other tissue-based productscomprised of terminally sterilized, non-viable cells which are incapableof completing the vascularization process. Further, in some aspects, theproduct of the present disclosure is not devitalized, or “fixed” withglutaraldehydes or radiation treatment.

In yet other aspects, the xenotransplantation products described anddisclosed herein are minimally manipulated (e.g., without physicalalteration of the related cells, organs or tissues) such that suchproducts are substantially in their natural state.

In certain aspects, the xenotransplantation products described anddisclosed herein are obtained from a non-human animal, e.g., anon-transgenic genetically reprogrammed swine, including cells, tissue,and/or an organ isolated from the non-transgenic geneticallyreprogrammed swine, the non-transgenic genetically reprogrammed swinecomprising a nuclear genome that has been reprogrammed to replace aplurality of nucleotides in a plurality of exon regions of a majorhistocompatibility complex of a wild-type swine with nucleotides fromorthologous exon regions of a known human major histocompatibilitycomplex sequence from a human capture sequence, and wherein cells ofsaid genetically reprogrammed swine do not present one or more surfaceglycan epitopes, wherein said reprogramming does not introduce anyframeshifts or frame disruptions. For example, genes encoding alpha-1,3galactosyltransferase, cytidine monophosphate-N-acetylneuraminic acidhydroxylase (CMAH), and β1,4-N-acetylgalactosaminyltransferase aredisrupted such that surface glycan epitopes encoded by said genes arenot expressed. Further specific aspects, details and examples areprovided in the following disclosures and claims and any and allcombinations of those aspects, details and examples constitute aspectsof the present disclosure.

In yet other aspects, the xenotransplantation products described anddisclosed herein are capable of making an organic union with the humanrecipient, including, but not limited to, being compatible withvascularization, collagen growth (e.g., in regard to skin), and/or otherinteractions from the transplant recipient inducing graft adherence,organic union, or other temporary or permanent acceptance by therecipient.

In yet other aspects, the xenotransplantation products described anddisclosed herein are utilized in xenotransplantation without the need touse, or at least reduction of use, of immunosuppressant drugs or otherimmunosuppressant therapies to achieve desired therapeutic results.

In other aspects, some of the xenotransplantation products described anddisclosed herein (e.g., skin) are stored by cryopreservation, storedfresh (without freezing), or stored via other methods to preserve suchproducts consistent with this invention. Storage involves usingconditions and processes that preserve cell and tissue viability.

In some aspects, storage may involve storing organs, tissues, or cells,in any combination of a sterile isotonic solution (e.g., sterile salinewith or without antibiotics), on ice, in a cryopreservation fluid,cryopreserved at a temperature of around −40° C. or around −80° C., andother methods known in the field. Such storage can occur in a primarycontainment system and secondary containment system.

In yet other aspects, the xenotransplantation products described anddisclosed herein are for homologous use, i.e., the repair,reconstruction, replacement or supplementation of a recipient's organ,cell and/or tissue with a corresponding organ, cell and/or tissue thatperforms the same basic function or functions as the donor (e.g., swinekidney is used as a transplant for human kidney, swine liver is used asa transplant for human liver, swine skin is used as a transplant forhuman skin, swine nerve is used as a transplant for human nerve and soforth).

In yet other aspects, the xenotransplantation products described anddisclosed herein have a low bioburden, minimizing pathogens, antibodies,genetic markers, and other characteristics that may serve to increasethe product's bioburden and the human body's immunological rejection ofthe product upon xenotransplantation. This may include the innate immunesystem, through PRRs TLRs, detecting PAMPs and rejecting the subjectxenotransplantation product.

It will be understood that the aspects disclosed and described hereincan be applied in any number of combinations to create an array ordifferent aspects comprising one or more of the features and/or aspectsof the aspects encompassed by the present invention.

It will be understood that there are numerous therapeutic applicationsfor products derived from DPF Closed Colony in accordance with thepresent invention. For example, such products may be utilized to treatacute and/or chronic disease, disorders, or injuries to organ, cells ortissue, and any and all other ailments that can utilize the productsdisclosed herein. Such treatments and/or therapies can include utilizingsuch products to repair, reconstruct, replace or supplement (in someaspects on a temporary basis and in other aspects a permanent basis), ahuman recipient's corresponding organ, cell and/or tissue that performsthe same basic function or functions as the donor.

Specific treatment applications include, but are not limited to, lungtransplants, liver transplants, kidney transplants, pancreastransplants, heart transplants, nerve transplants and other full orpartial transplants. With regard to skin, treatment applications alsoinclude, but are not limited to, treatment of burn wounds, diabeticulcerations, venous ulcerations, chronic skin conditions, and other skinailments, injuries and/or conditions (including, but not limited to,severe and extensive, deep partial and full thickness injuries, ailmentsand/or conditions) (see, e.g., Example 2 herein); use in adult andpediatric patients who have deep dermal or full thickness burnscomprising a total body surface area greater than or equal to 30%,optionally in conjunction with split-thickness autografts, or alone inpatients for whom split-thickness autografts may not be an option due tothe severity and extent of their wounds/burns; treatment of liverfailure, wounds, ailments, injuries and/or conditions with liverproducts derived in accordance with the present invention; treatment ofperipheral nerve damage, and other nerve ailments, injuries and/orconditions; and cell and other therapies utilizing materials harvestedfrom the DPF Closed Colony, including the therapeutic uses disclosed inU.S. Pat. No. 7,795,493 (“Phelps”), including cell therapies and/orinfusion for certain disorders (as disclosed in col. 30, line 1 to col.31, line 9) and treatment or certain disorders or pathologies (asdisclosed in col. 31, lines 10 to 42), the disclosure of which isincorporated by reference herein.

It will be understood that the specific recitation of therapies hereinin no way limits the types of therapeutic applications for the productsdisclosed and described herein, which encompass acute and/or chronicdisease, disorders, injuries to the following organs, tissues and/orcells: skin, kidneys, liver, brain, adrenal glands, anus, bladder,blood, blood vessels, bones, brain, brain, cartilage, ears, esophagus,eye, glands, gums, hair, heart, hypothalamus, intestines, largeintestine, ligaments, lips, lungs, lymph, lymph nodes and lymph vessels,mammary glands, mouth, nails, nose, ovaries, oviducts, pancreas, penis,pharynx, pituitary, pylorus, rectum, salivary glands, seminal vesicles,skeletal muscles, skin, small intestine, smooth muscles, spinal cord,spleen, stomach, suprarenal capsule, teeth, tendons, testes, thymusgland, thyroid gland, tongue, tonsils, trachea, ureters, urethra,uterus, uterus, vagina, areolar, blood, adenoid, bone, brown adipose,cancellous, cartaginous, cartilage, cavernous, chondroid, chromaffin,connective tissue, dartoic, elastic, epithelial, Epithelium, fatty,fibrohyaline, fibrous, Gamgee, Gelatinous, Granulation, gut-associatedlymphoid, Haller's vascular, hard hemopoietic, indifferent,interstitial, investing, islet, lymphatic, lymphoid, mesenchymal,mesonephric, mucous connective, multilocular adipose, muscle, myeloid,nasion soft, nephrogenic, nerve, nodal, osseous, osteogenic, osteoid,periapical, reticular, retiform, rubber, skeletal muscle, smooth muscle,and subcutaneous tissue; blood cells, blood precursor cells, cardiacmuscle cells, chondrocytes, cumulus cells, endothelial cells, epidermalcells, epithelial cells, fibroblast cells, granulosa cells,hematopoietic cells, Islets of Langerhans cells, keratinocytes,lymphocytes (B and T), macrophages, melanocytes, monocytes, mononuclearcells, neural cells, other muscle cells, pancreatic alpha-1 cells,pancreatic alpha-2 cells, pancreatic beta cells, pancreatic insulinsecreting cells, adipocytes, epithelial cells, aortic endothelial cells,aortic smooth muscle cells, astrocytes, basophils, bone cells, boneprecursor cells, cardiac myocytes, chondrocytes, eosinophils,erythrocytes, fibroblasts, glial cells, hepatocytes, keratinocytes,Kupffer cells, liver stellate cells, lymphocytes, microvascularendothelial cells, monocytes, neuronal stem cells, neurons, neutrophils,pancreatic islet cells, parathyroid cells, parotid cells, platelets,primordial stem cells, Schwann cells, smooth muscle cells, thyroidcells, tumor cells, umbilical vein endothelial cells, adrenal cells,antigen presenting cells, B cells, bladder cells, cervical cells, conecells, egg cells, epithelial cells, germ cells, hair cells, heart cells,kidney cells, leydig cells, lutein cells, macrophages, memory cells,muscle cells, ovarian cells, pacemaker cells, peritubular cells,pituitary cells, plasma cells, prostate cells, red blood cells, retinalcells, rod cells, Sertoli cells, somatic cells, sperm cells, spleencells, T cells, testicular cells, uterine cells, vaginal epithelialcells, white blood cells, ciliated cells, columnar epithelial cells,dopaminergic cells, dopaminergic cells, embryonic stem cells,endometrial cells, fibroblasts fetal fibroblasts, follicle cells, gobletcells, keratinized epithelial cells, lung cells, mammary cells, mucouscells, non-keratinized epithelial cells, osteoblasts, osteoclasts,osteocytes, and squamous epithelial cells. This listing is in no waymeant to limit the array of therapeutic uses to treat acute and/orchronic disease, disorders, injuries, organ or tissue failures, and anyand all other ailments that can utilize the products disclosed herein.

With respect to the treatment of burns, including but not limited toe.g., second- and third-degree burns, in some aspects, skin productsderived in accordance with the present invention are used to treat humanpatients with severe and extensive deep partial and/or full thicknessburn wounds. Such products contain terminally-differentiated cell typesthat are not expanded ex vivo prior to use and do not migrate from thesite of application during intended duration of treatment. Therefore,potential for tumorigenicity is negligible.

Such products adhere to the wound bed and provides a barrier function inthe immediate post-burn period. Such products have non-terminallysterilized, viable cells, allowing for vascularization of the grafttissue with the recipient. In some aspects, the epidermis remains fullyintact, and dermal components are maintained without change tostructural morphology or organization of the various cells and tissues.This physiologic mechanism supports the prolonged survival of the graftmaterial, and provides at least a temporary barrier function withsignificant clinical impact on par with, or better than, allograft. Insome aspects, if clinical signs of infection, e.g., pain, edema,erythema, warmth, drainage, odor or unexplained fever, are present ordeveloping, the product of the present disclosure is not applied untilthe clinical signs of the infection are reduced or eliminated for apredetermined period of time, e.g., 1, 2, 3, 4, 5, 6, or 7 days, 1, 2,3, or 4 weeks, or if the subject has tested negative for the infection.In some aspects, the wound is cleaned, confirmed to be well-vascularizedand nonexuding. If a dermal substitute such as cadaver allograft is alsobeing used, the epidermal layer is removed from engrafted allograftprior to the application of the product without removing the engrafteddermis. The epidermal layer may be removed with a dermatome or otherinstrument according to standard operating procedures of the facility.

Grafts conventionally used in clinical practice consist ofdecellularized and/or reconstituted sheets of homogenized dermis thatare used to achieve temporary, superficial wound coverage. Suchconventional grafts do not retain the original tissue structure nor themetabolically active, otherwise naturally present cells, and thus do notbecome vascularized; no capillary ingrowth or vessel-to-vesselconnections are made. In contrast, skin products described herein arefundamentally differentiated from such grafts because the product of thepresent disclosure includes live cells that perform the same function asthe patient's original skin, i.e., the product acts as an organtransplant. Skin performs additional, critical roles related tohomeostasis, temperature regulation, fluid exchange, and infectionprevention. The absence of a sufficient amount of skin can compromisethe ability to perform these functions leading to high incidences ofmortality and morbidity from infections and fluid loss. Skin transplantshave been reliably used with notable clinical benefit to prevent theseoutcomes in patients with significant wounds; regardless of whether thegraft is temporary or permanent. Thus, unlike other proposedtransplants, use of immunosuppressive drugs would be reduced or not benecessary. In fact, such regimens would be contraindicated in burnpatients whose injuries already exhibit some level of comprised immunefunction. Thus, the xenotransplantation product of the presentdisclosure should not be confused with traditional “xenograft” productsconsisting of econstituted, homogenized wild-type porcine dermisfashioned into sheets or meshed, such as EZ-Derm™ or Medi-Skin™. Suchporcine xenografts do not vascularize and are primarily only useful fortemporary coverage of superficial burns. In stark contrast, thexenotransplantation product of the present disclosure containsmetabolically active, minimally manipulated cells in identicalconformations and unchanged morphologies as the source tissue.

In some aspects, the present disclosure includes using xenotransplanteddonor skin as a test for prediction of rejection of other organs fromthe same animal donor. Techniques for performing such predictive testsusing human donor skin have previously been described, e.g., in Moraeset al., Transplantation. 1989; 48(6):951-2; Starzl, et al., Clinical andDevelopmental Immunology, vol. 2013, Article ID 402980, 1-9; Roberto etal., Shackman et al., Lancet. 1975; 2(7934):521-4, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes. Moraes reported that the crossmatch procedure was highlyaccurate in predicting early kidney transplant rejection. Shackmanreported that the fate of skin grafts taken from live human prospectivekidney donors correlates well with the outcome of kidney transplantationfrom the same donors. According to the present disclosure, in oneaspect, the present disclosure includes a method of using axenotransplanted skin sample in a human patient in order to determinewhether there is a risk of rejection of other organs xenotransplantedfrom the same animal donor in the human patient.

The skin grafting methods described herein can be used to treat anyinjury for which skin grafts are useful, e.g., for coverage of partialthickness and full thickness wounds including but not limited to burns,e.g. partial thickness or excised full thickness burn wounds; avulsedskin e.g. on an extremity; diabetic wounds, e.g., non-healing diabeticfoot wounds, venous stasis ulcers.

In some aspects, the xenotransplantation product of the presentdisclosure has pharmacokinetic and pharmacodynamics properties that meetregulatory requirements. Characterization of such properties requires aunique approach with respect to classical meanings of drug absorption,distribution, metabolism, and excretion. “Absorption” of thexenotransplantation product for the purposes of consideration ofpharmacokinetics, may be described by the vascularization process thexenotransplantation product experiences. For example, shortly aftersurgery, skin xenotransplantation products may present as warm, soft,and pink, whereas wild-type or traditional xenografts appear asnon-vascularized “white grafts.” In some aspects, the distribution ofthe transplant is limited to the site of transplant as confirmed by DNAPCR testing to demonstrate the presence or absence of pig cells inperipheral blood beyond the transplantation site.

In other aspects, the cells of the biological products produced inaccordance with the present invention do not migrate followingxenotransplantation into the recipient, including into the circulationof the recipient. This includes that PERV or PERV-infected porcine cellsdo not migrate into the recipient. Confirmation that such cells do notmigrate into the recipient can be performed in a number of ways,including via DNA-PCR analysis of peripheral blood mononuclear cells(PBMCs) and samples from the transplantation site and of highly perfusedorgans (e.g., liver, lung, kidney and spleen) to determine and otherwisedemonstrate that migrations of porcine cells (DNA) or porcine retroviral(RNA) components in the peripheral blood did not occur in the recipient.

Moreover, bioavailability and mechanism of action of thexenotransplantation product is not affected by size. The distribution ofthe xenotransplantation product is limited to the site of theadministration. For example, in the case of a skin transplant, thedebrided wound bed initially created by the trauma or burn injury is thesite of administration. The present disclosure includes testing todetect distribution of cells from the xenotransplantation product in theperipheral blood, wound beds, spleen and/or kidney beyond the site ofadministration. In certain aspects, such testing will demonstrate anabsence of cells from the xenotransplantation product in the peripheralblood, wound beds, spleen and/or kidney beyond the site ofadministration. Such testing may include DNA PCR testing for variouscellular markers present in the type of animal from which the product isobtained, e.g., PERV, swine MHC, and other swine DNA sequences. Incertain aspects, cells and nucleic acids from the xenotransplantationproduct remain limited to the site of administration.

The metabolism of the xenotransplantation product, traditionally definedas the metabolic breakdown of the drug by living organisms, typicallyvia specialized enzymes or enzymatic systems, may be congruent with theaforementioned natural host rejection phenomenon, which occurs in theabsence of exogenous immunosuppressive drugs. Via the same formulationand identical route of administration as intended for future human use,such xenotransplantation products undergo a delayed, immune rejectioncourse similar to allograft comparators for clinically useful durations.

In similar fashion, excretion of the xenotransplantation product couldbe modeled and experientially monitored by the clinical “sloughing”phenomenon as a result of necrotic ischemia of the transplant, due toantibody-mediated vascular injury, ultimately leading to the death ofthe tissue.

The demonstrated efficacy of the xenotransplantation product of thepresent disclosure, along with safety, availability, storage,shelf-life, and distribution, provide significant advantages overcurrent standards of care.

In some aspects, the “dosage” of the xenotransplantation product of thepresent disclosure is expressed as percentage of viable cells in theproduct per unit area of transplantation. As such, in some aspects, thexenotransplantation product of the present disclosure can be consideredas analogous to the active pharmaceutical ingredient in a pharmaceuticaldrug product.

Survival of the xenogeneic cells, tissues, or organs of the presentdisclosure is increased by avoiding: (a) infiltration of immune orinflammatory cells into the xenotransplantation product or alteration ofsuch cells in other relevant compartments, such as the blood andcerebrospinal fluid; (b) fibrotic encapsulation of thexenotransplantation product, e.g., resulting in impaired function orxenotransplantation product loss; (c) xenotransplantation productnecrosis; (d) graft versus host disease (GVHD); and (e) in vivo functionand durability of encapsulation or barriers intended to diminishrejection or inflammatory responses.

Blood samples from piglets are obtained and tested for phenotype, lackof expression of alpha galactose on the cell surface of blood cellsusing FITC-IB4 labeling and flow cytometry. At this stage ofdevelopment, all progeny will be genotyped at birth. A PCR assay hasbeen established to determine if a pig has a wild typegalactose-α1,3galactose transferase gene (Gal-T) or if it isheterozygous or homozygous for the Gal-T knockout (Gal-T-KO) using DNAisolated from ear notches or PBMC. Genomic DNA is isolated from PBMC (orskin tissues) using DNeasy Kit following the Qiagen DNeasy kitdirections. PCR is performed on genomic DNA and control template DNA,Wild type Gal-T (+/+) Heterozygote Gal-T-KO (+/−) and HomozygousGal-T-KO (−/−).

Punch biopsies of skin grafts are co-cultured with subconfluent targetcells, human 293 (kidney epithelium) and porcine ST-IOWA cell linesmaintained in culture medium (Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum and glutamine, penicillin, andstreptomycin) in a 75-cm2 flask. The biopsies are kept in contact withthe target cells for 5 days, after which the culture medium andremaining tissue are removed and the target cell co-cultures aremaintained by subculturing as necessary. PERV infection of target cellsis determined by the presence of reverse transcriptase (RT) activity inthe culture supernatants. Transmission assays are maintained for aminimum of 60 days before being considered negative.

Product characterization to measure safety, identity, purity and potencyis performed. Safety tests include bacterial and fungal sterility,Mycoplasma, and viral agents. The present disclosure includescryopreserving and archiving for further testing, as needed, samples ofall final xenotransplantation products (i.e., cells or tissues orbiopsies of organs), whether fresh or from culture ex vivo. In somecases, for example if the xenotransplantation product is a whole intactorgan, a relevant surrogate sample (e.g., adjacent tissues orcontra-lateral organ) is archived.

With regard to skin, storage and cryopreservation of porcine skin hasnot been fully characterized, especially with regards to viability, asmost porcine xenografts are intentionally devitalized, or “fixed” withglutaraldehydes or radiation treatment. Such information is necessary tosupport the use of vital porcine skin grafts—or porcine skintransplants—as a temporary and clinically advantageous option.

In procedures in which the xenotransplantation product is transplantedimmediately after removal from the source animal, such asxenotransplantation of whole organs, results of testing of thexenotransplantation product may not be available before its clinicaluse. In such cases, testing of the source animal, itself, may be all thetesting that is possible before the procedure. Testing of samples takenfrom such xenotransplantation products or appropriate relevantbiological surrogates, e.g., adjacent tissues or contra-lateral organs,may be performed according to the present disclosure. Microbiologicalexamination methods may include aspects set forth in the following Table2:

TABLE 2 SUITABILITY OF COUNTING METHOD IN GROWTH THE PRESENCE OFPROMOTION PRODUCT TEST DETAILS Total Total Total Preparation AerobicYeasts and Total Aerobic Yeasts and of Test Microbial Molds MicrobialMolds Microorganism Strain Count Count Count Count StaphylococcusSoybean- Soybean- Soybean-Casein aureus such Casein Digest Casein Digestas ATCC Agar or Digest Agar Agar/MPN 6538, NCIMB Soybean- and Soy-Soybean-Casein 9518, CIP Casein Digest bean-Casein Digest Broth 4.83, orBroth Digest Broth ≤100 cfu NBRC 30°-35° ≤100 cfu 30°-35° 13276 18-24hours 30°-35° ≤3 days ≤3 days Pseudomonas Soybean- Soybean-Soybean-Casein aeruginosa Casein Digest Casein Digest such as ATCC Agaror Digest Agar Agar/MPN 9027, NCIMB Soybean- and Soy- Soybean-Casein8626, CIP Casein Digest bean-Casein Digest Broth 82.118, Broth DigestBroth ≤100 cfu or NBRC 1 30°-35° ≤100 cfu 30°-35° 3275 18-24 hours30°-35° ≤3 days ≤3 days Bacillus Soybean- Soybean- Soybean-Caseinsubtilis such Casein Digest Casein Digest as ATCC Agar or Digest AgarAgar/MPN 6633, NCIMB Soybean- and Soy- Soybean-Casein 8054, CIP CaseinDigest bean-Casein Digest Broth 52.62, or Broth Digest Broth ≤100 cfuNBRC 3134 30°-35° ≤100 cfu 30°-35° 18-24 hours 30°-35° ≤3 days ≤3 daysCandida Sabouraud Soybean- Sabouraud Soybean-Casein Sabouraud albicanssuch Dextrose Agar Casein Dextrose Digest Agar Dextrose as ATCC orSabouraud Digest Agar ≤100 cfu ≤100 cfu Agar 10231, NCPF Dextrose ≤100cfu 20°-25° 30°-35° ≤100 cfu 3179, Broth 20°-25° 30°-35° ≤5 days ≤5 days20°-25° IP 48.72, or 2-3 days ≤5 days MPN: not ≤5 days NBRC 1594applicable Aspergillus Sabouraud Soybean- Sabouraud Soybean-CaseinSabouraud brasiiiensis Dextrose Casein Dextrose Digest Agar Dextrosesuch as Agar or Digest Agar ≤100 cfu ≤100 cfu Agar ATCC16404, Potato-≤100 cfu 20°-25° 30°-35° ≤100 cfu IMI 149007, Dextrose 30°-35° ≤5 days≤5 days 20°-25° IP 1431.83, or Agar 20°-25° ≤5 days MPN: not ≤5 daysNBRC 9455 5-7 days, or applicable until good sporulation is achieved

The present disclosure includes using Buffered Sodium Chloride-PeptoneSolution pH 7.0 or Phosphate Buffer Solution pH 7.2 to make testsuspensions; to suspend A. brasiliensis spores, 0.05% of polysorbate 80may be added to the buffer. The present disclosure includes using thesuspensions within 2 hours, or within 24 hours if stored between 2° C.and 8° C. As an alternative to preparing and then diluting a freshsuspension of vegetative cells of A. brasiliensis or B. subtilis, astable spore suspension is prepared and then an appropriate volume ofthe spore suspension is used for test inoculation. The stable sporesuspension may be maintained at 2° to 8° for a validated period of time.To verify testing conditions, a negative control is performed using thechosen diluent in place of the test preparation. There must be no growthof microorganisms. A negative control is also performed when testing theproducts as described under Testing of Products. A failed negativecontrol requires an investigation. Microbiological Examination may beperformed according to USP 61, USP 63, USP 71, USP 85 EP section 2.6.13Microbial Examination of Non-sterile Products (Test for SpecifiedMicroorganisms), each of which is incorporated herein by reference inits entirety.

With regard to testing for porcine cytomegalovirus (PCMV), sourceanimals are screened for PCMV on a quarterly basis. However, caesarianderived piglets, which are then consistently raised in the closed colonyare not infected with PCMV. Analysis for PCMV was conducted during thestudies in Example 1 herein and no PCMV was detected in the punchbiopsies using the following PCR method. These results were consistentto the PCR results from nasal swabs. Quantitative Real-Time PCR isutilized for PCMV testing. Target DNA sequences were quantified byreal-time PCR using a Stratagene Mx3005P. Sequence-specific primers andTaqMan probe were generated for each gene target. Each 25 uL PCRreaction included target DNA, 800 nM primers 200 nM TaqMan probe, 20 nMRox reference and 1× Brilliant III Ultra Fast Master Mix. The PCRcycling conditions were as follows: 1 cycle at 95° C. for 5 min followedby 50 cycles of denaturation at 95° C. for 10 seconds, andannealing-extension at 60° C. for 30 seconds with data collectionfollowing each extension. Serial dilutions of gel-extracted ampliconcloned into Invitrogen TOPO plasmid served as quantifying standards.Target DNA is detected with a linear dynamic range of 10 to 106 copies.For quantification of PCMV DNA, 300 ng of xenograft pig kidney DNA wasrun in a TaqMan PCR in triplicate. Primers and probes specific for PCMVDNA polymerase gene have been shown to have no cross-reactivity withPLHV-1. Utilization of cesarean-derived swine as source animals,combined with animal husbandry of the resulting closed colony andmaintenance of the barrier-isolation conditions is attributed theanimals being PCMV free. With regard to skin, the inventors noted thatthe safety and efficacy results achieved in Example 1 using singleknockout swine (as opposed to triple knockout or even furthergenetically modified swine) were quite surprising given the comparableperformance to allograft.

In some aspects, the analytical procedures used to test thexenotransplantation product can also include:

a. USP<71> Sterility.

Samples are transferred to Tryptic Soy Broth (TSB) or FluidThioglycollate Medium (FTM) as appropriate. For Bacteriostasis andfungistasis, TSB samples are spiked with an inoculum of <100 ColonyForming Units (CFUs) of 24-hour cultures of Bactillus subtilis, Candidaalbicans, and with <100 spores of Aspergilius braseiliensis. The FTMsamples will be spiked with an inoculum of <100 CFU's of 24-hourcultures of Staphyloccocus aureus, Pseudomonas aeruginosa, andClostridium sporogenes. If growth is not observed, the product is foundto be bacteriostatic or fungistatic and fails the USP <71> SterilityTest.

b. Aerobic and Anaerobic Bacteriological Cultures.

Samples are transferred to Tryptic Soy Broth (TSB) or FluidThioglycollate Medium (FTM) as appropriate. Vessels will be incubated toallow for potential growth. If no evidence of microbial growth is found,the product will be judged to comply with the test for sterility asdescribed by USP<71>.

c. Mycoplasma Assay USP <63>.

Fresh samples will be added to 100 mL of Mycoplasma Hayflick broth andincubated at 37° C. for up to 21 days. The sample is subcultured after2-4 days, 7-10 days, 14 days, and 21 days. The plates are then incubatedat 37° C. for up to 14 days and checked for the presence of Mycoplasmacolonies. If none are detected, the product is found to be in compliancewith USP<63> and is Mycoplasma free.

d. Endotoxin USP<85>.

Three samples from the same lot will be tested for theInhibition/Enhancement of the Limulus amoebocyte lysate (LAL) test.Samples will be extracted with 40 mL of WFI per sample at 37° C. for 1hour. Samples will then be tested in the LAL Kinetic Chromogenic Testwith a standard curve ranging from 5-50 EU/mL at a validated dilution.Assays will be performed in compliance with USP<85>.

e. MTT Assay for Cell Viability.

The metabolic activity of the drug product is tested relative to controltissue samples using a biochemical assay for [3-4,5dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) metabolism.Positive and negative control samples of fresh xenotransplantationproduct tissue (positive control) or heat inactivated discs ofxenotransplantation product tissue (negative control) or the testarticle of Xenotransplantation product are placed in ambermicrocentrifuge tubes containing MTT solution (0.3 m g/mL in DMEM, 0.5mL). The discs are treated with MTT formazan and incubated for 180±15minutes at 37° C. and an atmosphere of 5% CO 2 in air. The reaction isterminated by removal of the discs and the formazan is extracted byincubation at either ambient temperature for ≤24 hours or refrigeratedat 4° C. for ≤72 hours. Samples are protected from light during thistime. Aliquots are taken after the extraction is complete and theabsorbance at 550 nm (with a reference wavelength of 630 nm) is measuredand compared to a standard curve.

f. IB4 Assay for Extracellular Glycan Epitope.

The absence of the galactosyl-α-1,3-galactose (Alpha-Gal) epitope oncells will be determined using fluorescence activated flow cytometry.White blood cells in whole blood are stained with a fluorochrome labeledisolectin-B4 (FITC-I-B4) and comparisons are made against blood obtainedfrom wild type positive controls and the Gal-T-KO source animal twice.First, all source animals are tested at birth. Second, the same testwill be performed from whole blood collected at sacrifice of the sourceanimal and tested for stability of the gene knockout, and the negativephenotype for Alpha-Gal. The isolectin binds to the epitope on cellsfrom the wild type pig but no binding occurs on the cells from theGal-T-KO pigs. The assay serves to confirm alpha-gal epitope is notpresent in the genetically engineered source animal. Spontaneousre-activation of the gene, and re-expression of the Alpha-Gal moietypost sacrifice is highly improbable and unreasonable to expect; itsinclusion would only deteriorate the efficacy of the xenotransplantationproduct causing it to resemble wild-type porcine tissue and hyperacutelyreject as previously demonstrated.

g. PERV Viral Assay.

PERV pol quantitation 10 uL of a 1:625 dilution of the RT reaction wasamplified in a 50 cycle PERV polymerase quantitative TaqMan PCR intriplicate using a Stratagene MX300P real-time thermocycler (AgilentTechnologies). 10 uL of a 1:25 dilution of the “No RT enzyme” control RTreaction was similarly treated. PCR conditions included PERV pol forwardand reverse primers at 800 nM final concentration and PERV pol probe at200 nM final concentration. Brilliant III Ultra Fast master mix (600880Agilent Technologies) was used supplemented to 20 nM with ROX reporterdye (600880 Agilent Technologies) and 0.04 Units/μL UNG nuclease(N8080096, Life Technologies). Cycling conditions included 1 cycle of 10minutes at 50° C. followed by one cycle of 10 minutes at 95° C. and 50cycles of 10 seconds at 95° C. followed by 30 seconds at 60° C. withdata collected at the end of each cycle. Absolute copies of PERV pol,and of porcine MHC-I and porcine GAPDH nucleic acids were measured pernanogram of input cDNA. Punch biopsies of thawed as described herein andwashed xenotransplantation product are tested for the presence of PERVDNA and RNA.

h. Histology and Morphology.

Samples of the xenotransplantation product, following the describedmanufacturing process, are sampled for examination for cell morphologyand organization. Verification under microscope via visible examinationto ensure correct cell morphology and organization ofxenotransplantation product tissues and absent for abnormal cellinfiltrate populations.

i. Release Assay Sampling Methodology.

Once all units of the final xenotransplantation product lot have beencreated, units are independently, randomly selected for use inmanufacturing release assays for the required acceptance criteria. Theseunits will be marked for lot release to the various laboratorycontractors, and the various analytical tests will be performed per therequired cGMP conditions.

Similarly, prior to validation for human clinical use, all finalxenotransplantation product must meet acceptance criteria for selectinga donor pig for material including (i) reviewing the medical record fora defined pedigree, (ii) reviewing the medical record for the testresults for alpha-1,3-galactose by Flowmetrics, (iii) reviewing themedical record for a history of full vaccinations; (iv) reviewing themedical record for the surveillance tests performed over the lifetime ofthe pig; (v) adventitious agent screening of source animal; (vi)reviewing the medical record for infections over the lifetime of thepig; and (vi) reviewing the medical record for any skin abnormalitiesnoted in the animal's history.

The final xenotransplantation product control strategy and analyticaltesting is conducted at the conclusion of the manufacturing processprior to release for clinical use. Results of the required analyticaltests will be documented via a xenotransplantation product drug productCertificate of Analysis (COA) that is maintained with a master batchrecord pertaining to each lot of xenotransplantation product drugproduct.

The following Table 3 is a list of the assays and results of the batteryof tests performed on the xenotransplantation product materials.

TABLE 3 Sample Material Test Test Method Tested Result Sterility TestingTissue Culture 3 mm Punch No growth detected Aerobic Bacteria Biopsy ofAnaerobic Bacteria Xenotrans- Fungi plantation Acid fast culturesproduct Specific bacterial (Post Thaw) screen Mycological ScreenMycoplasma Assay 3 mm Punch No growth Biopsy of detected after 28Xenotrans- days plantation product (Post Thaw) Bacteriostasis & USP<71>Xenotrans- Bacteriostatic, no Fungistasis Gibraltar Laboratoryplantation growth of specific product indicator organism (Post Thaw)Endotoxin Test USP<85> LAL, Xenotrans- <0.2 EU/unit Kinetic plantationChromogenic Test product (Post Thaw) Endogenous Viral Testing RT-qPCR 3mm Punch Presence of (PERV) Co-culture Assay Biopsy of PERV A, B, MGH-Xenotrans- C Infectious plantation confirmed Disease- product Fishman(Post Thaw) Laboratory Viability Testing MTT and 3 mm Punch Greater than70% Phenyl Acetate Biopsy Mitochondrial Assays of Activity remainingXenotrans- following freeze-thaw plantation cycle, confirmed by productboth assays (Post Thaw) Identity Histology, 3 mm Punch No abnormalitiesCell Morphology Hematoxylin and Biopsy noted. Eosin Staining of Cellmorphology Xenotranspl and organization antation consistent with skinproduct graft (Post Thaw) No presence of Alpha-GAL detected ConfirmationFlow Cytometry, Whole Blood, 2 of absence of isolectin-B4 (FITC- ml,obtained Alpha-GAL I-B4) from source (Gal-T- animal, at Knockoutsacrifice. confirmation)

In another aspect it will be understood that there includes anadventitious agent control strategy developed based on the sourceanimal, including the species, strain, geographic origin, type oftissue, and proposed indication. Analytical Tests are conducted foradventitious agents, to include bacteria, fungi, Mycoplasma, and viralmicroorganisms, including as follows:

j. Bacteriological Free Status—

The bacteriological screen is conducted to confirm the drug product isfree of potential biological agents of concern Humans. Both Aerobic andAnaerobic screens are conducted to ensure sterility. Samples are thawedas described herein and transferred to Tryptic Soy Broth (TSB) or FluidThioglycollate Medium (FTM) as appropriate. Vessels will be incubated toallow for potential growth. If no evidence of microbial growth is found,the product will be judged to comply with the test for sterility.

k. Mycological (Fungal) Free Status—

The mycological screen is conducted to confirm the Drug Product is freeof potential fungal agents of concern. Samples are thawed as describedherein. After thawing, samples are transferred to a soybean-caseindigest agar. Vessels will be incubated to allow for potential growth. Ifno evidence of fungal growth is found, the product will be judged tocomply with the test for sterility per USP<71>.

l. Mycoplasma Free Status—

The Mycoplasma screen is conducted to confirm the drug product is freeof Mycoplasma. Samples are thawed as described herein and added to 100mL of Mycoplasma broth and incubated at 37° C. for up to 21 days. Thesample is sub-cultured after 2-4 days, 7-10 days, 14 days, and 21 days.The plates are then incubated at 37° C. for up to 14 days and checkedfor the presence of Mycoplasma colonies. If none are detected, theproduct is found to be in compliance with USP<63> and is Mycoplasmafree.

m. Endotoxin Free Status—

The endotoxin free status is conducted to confirm the drug product isfree of endotoxins and related agents of concern. Three samples from thesame lot will be tested for the Inhibition/Enhancement of the Limulusamoebocyte lysate (LAL) test. Samples will be thawed as described hereinand extracted with 40 mL of WFI per sample at 37° C. for 1 hour. Sampleswill then be tested in the LAL Kinetic Chromogenic Test with a standardcurve ranging from 5-50 EU/mL at a validated dilution. Assays will beperformed in compliance with USP<85>.

n. Viral Assays Conducted—

The viral assays are conducted to confirm the source animal is free ofpotential viral agents of concern, confirmation of endogenous viruses(see below). This includes co-culturing and RT-PCR testing for specificlatent endogenous viruses including PERV. In vivo assays are alsoconducted on the animal source to monitor animal health and freedom fromviral infection as key aspects of the lot release criteria. Due to theendemic nature of PERV in porcine tissue, this qualifies as a positiveresult that does not preclude the use of such tissue. However, the virusis identified and characterized in lot release to provide informationfor monitoring the recipient of the xenotransplantation product.

o. Cell Viability Assay—

The MTT assay is conducted to confirm the biologically active status ofcells in the xenotransplantation product. Evidence of viability isprovided through surrogate markers of mitochondrial activity as comparedto positive (fresh, not cryopreserved) and negative (heat-denatured)controls. The activity of the cells is required for thexenotransplantation product to afford the intended clinical function.This is required as a lot release criteria, and is currently establishedthat tissue viability should not be less than 50% of the metabolicactivity demonstrated by the fresh tissue control comparator.

p. Histology and Morphology—

Verification under microscope via visible examination of Hematoxylin andEosin (H&E) section staining of the epidermal and dermal layers, toensure correct cell morphology and organization of thexenotransplantation product tissues and cell infiltrate populations.This is conducted to confirm the appropriate physiologic appearance andidentity of cells present in the xenotransplantation product. Thexenotransplantation product is composed of minimally manipulated porcinedermal and epidermal tissue layers. This is required as a lot releasecriteria. Evidence of the following cell layers (from most superficialto deepest), in the epidermal layer are verified:

-   -   i. Stratum Corneum    -   ii. Stratum Granulosum    -   iii. Stratum Spinosum    -   iv. Stratum Basale        Evidence of the following cellular structures in the dermal        layer are verified:    -   v. Blood vessels, evidence of vasculature    -   vi. Nerves    -   vii. Various glands    -   viii. Hair follicles    -   ix. Collagen

The genetically engineered source animals do not contain any foreign,introduced DNA into the genome; the gene modification employed isexclusively a knock-out of a single gene that was responsible forencoding for an enzyme that causes ubiquitous expression of acell-surface antigen. It will be understood that the xenotransplantationproduct in one or more aspects do not incorporate transgenetechnologies, such as CD-46 or CD-55 transgenic constructs.

An endotoxin free status is conducted to confirm the drug product isfree of endotoxins and related agents of concern. Protocols for theassurance of Endotoxin free status are as follows: Three samples fromthe same lot are tested for Inhibition/Enhancement of the Limulusamoebocyte lysate (LAL) test. Samples are thawed, extracted, and testedin the LAL Kinetic Chromogenic Test with a standard curve ranging from5-50 EU/mL at a validated dilution in compliance with USP<85>.

The MTT assay is conducted to confirm the biologically active status ofcells in the product. Evidence of viability is provided throughsurrogate markers of mitochondrial activity as compared to positive(fresh, not cryopreserved) and negative (heat-denatured) controls. Theactivity of the cells is required for the product to afford the intendedclinical function and the viability parameters for one aspect rangingfrom 50% to 100% mitochondrial activity.

Verification under microscope via visible examination of Hematoxylin andEosin (H&E) section staining of the epidermal and dermal layers, toensure correct cell morphology and organization of thexenotransplantation product tissues and cell infiltrate populations.This is conducted to confirm the appropriate physiologic appearance andidentity of cells present in the product.

For skin xenotransplantation products, evidence of the following celllayers (from most superficial to deepest), in the epidermal layer areverified: Stratum Corneum; Stratum Granulosum; Stratum Spinosum; StratumBasale. Evidence of the following cellular structures in the dermallayer are verified: Blood vessels, evidence of vasculature; Nerves;Various glands; Hair follicles; Collagen.

The xenotransplantation product may be further processed to ensure thatit remains free of aerobic and anaerobic bacteria, fungi, viruses, andMycoplasma. Under sterile conditions in a laminar flow hood in a drugproduct processing suite using applicable aseptic techniques,immediately after, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 seconds, within10 seconds to 1 minute, within 1 minute to 1 hour, within 1 hour to 15hours, or within 15 hours to 24 hours following harvest, thexenotransplantation product is sterilized, e.g., using one or more of UVirradiation or an anti-microbial/anti-fungal. In one aspect, the productmay be placed into an anti-microbial/anti-fungal bath (“antipathogenbath”). The antipathogen bath may include: one or more anti-bacterialagents, e.g., ampicillin, ceftazidime, neomycin, streptomycin,chloramphenicol, cephalosporin, penicillin, tetracycline, vancomyocin,and the like; one or more anti-fungal agents, e.g., amphotericin-B,azoles, imidazoles, triazoles, thiazoles, candicidin, hamycin,natamycin, nystatin, rimocidin, allylamines, echinocandins, and thelike; and/or one or more anti-viral agents. The anti-pathogen bath mayinclude a carrier or medium as a diluent, e.g., RPMI-1640 medium. Insome aspects, the anti-pathogen bath may include at least 2anti-bacterial agents. In some aspects, the anti-pathogen bath mayinclude at least 2 anti-bacterial agents and at least one anti-fungalagent. In some aspects, the anti-pathogen bath may include at least fouragents. In some aspects, the anti-pathogen bath may include no more than4, 5, 6, 7, 8, 9, or 10 agents. In some aspects, the anti-pathogen bathmay include any combination of the foregoing.

The product may be sterilized using UV light sterilization. For example,the product is placed under the UV lamp for a desired period of time,e.g., 0.5, 1, 1.5, 2, 3, 4, 5, 6, minutes or more, then turned over tothe other side, and put under the UV lamp for the same or a differentperiod of time on opposite side. The time period for exposing a givensample to the UV may be varied based on the specific biological agentsor the types of biological agents to be sterilized, e.g., as shown inthe following Table 11 below. For example, the product may be sterilizedusing a UV lamp having a UV-C intensity of at least 100 uW/cm² for atleast 2 minutes and up to 15, 12, 10, 8, 6, 5, 4, 3, or 2.5 minutes, andturned over such that its opposite surface is exposed to the UV lamp forat least 2 minutes and up to 15, 12, 10, 8, 6, 5, 4, 3, or 2.5 minutesto obtain a UV-treated product; a UV-C dosage of at least 100,000 uWsec/cm² and up to 800,000, 700,000, 600,000, 500,000, 400,000, 300,000or 200,000 uW sec/cm²; a UV-C dosage of at least 200,000 uW sec/cm² andup to 800,000, 700,000, 600,000, 500,000, 400,000, or 300,000 uWsec/cm²; a UV lamp having a UV-C intensity of at least 100 uW/cm² for atleast 2 minutes and up to 15, 12, 10, 8, 6, 5, 4, 3, or 2.5 minutes.

Product processing occurs in a single, continuous, and self-contained,segregated manufacturing event that begins with the sacrifice of thesource animal through completion of the production of the final product.The animal is euthanized via captive bolt euthanasia, may be moved, ifnecessary, in a sterile, non-porous bag, to an operating room where theprocedure to harvest biological product from the source animal willoccur. All members of the operating team should be in full sterilesurgical gear, e.g., dressed in sterile dress to maintain designatedpathogen free conditions prior to receiving the source animal and insome instanced be double-gloved to minimize contamination, and surgicalareas and tools are sterilized. The source animal is removed from thebag and container in an aseptic fashion. The source animal is scrubbedby operating staff, e.g., for at least 1-10 minutes with antiseptic,e.g., Chlorhexidine, brushes over the entire area of the animal wherethe operation will occur, periodically pouring Chlorhexidine over thearea to ensure coverage. Surgical area(s) of the animal are scrubbedwith opened Betadine brushes and sterile water rinse over the entirearea of the animal where the operation will occur for, e.g., 1-10minutes.

In one aspect, with regard to skin, a full thickness skin graft wounddressing consisting of dermal tissue derived from a swine in accordancewith the present invention is used in conjunction or combination withcultured epidermal autografts to produce a product according to thepresent disclosure and that can be used in methods of the presentdisclosure. Prior to application of the epidermal autografts,significant debridement of wound bed is required to ensure an adequatesubstrate. To confirm a wound bed is ready for an epidermal autograft,apply the skin products described herein, e.g., biological skin productsderived from animals of the present disclosure to confirm adherence.Once adherence is confirmed, the temporary wound coverage product isremoved, and in some aspects, the wound bed is covered with a meshedautograft, and one or more cultured epidermal autograft products areplaced on top to close the gaps in the autograft mesh.

The debridement may include mechanical debridement, chemicaldebridement, enzymatic debridement, or a combination thereof. Mechanicaldebridement may include surgical excision, e.g., tangential excision toremove thin layers of dermis until healthy tissue is visualized, orfascial excision to remove the full thickness of dermis down to theunderlying fascia. Tangential excision allows less viable tissue to beremoved with the necrotic tissue, but typically results in higher bloodloss, is a larger physiologic stressor than fascial excision, and ismore likely to result in “incomplete” debridement, with some devitalizedtissue remaining in place. In fascial excision, blood loss and operativetime are minimized, but often a large amount of healthy tissue isremoved with the burned tissue. Debriding agents may include agentscapable of cleaning a burn wound by removing foreign material and deadtissue. Many such agents are known. In enzymatic debridement,collagenases or other proteolytic enzymes are employed that break downproteins of the extracellular matrix, allowing devitalized tissue to bewiped away without the need for surgery while preferably leaving healthytissue substantially intact. Enzymatic debridement involves theapplication of proteolytic and optionally other exogenous enzymes to awound surface to break down necrotic tissue. Enzymatic debridement maybe a relatively slow process, carried out over a period of a number ofweeks in combination with other topical preparations, soakings andrepeated dressings. Alternately, rapid enzymatic debridement can beaccomplished using multi-enzyme products, for example, those extractedfrom the stem of the pineapple plant, as disclosed for example in WO98/053850 and WO 2006/0006167, and as provided in the product marketedunder the trade name Debrase®. A procedure for enzymatic debridementgenerally utilizes an enzyme such as bromelain derivatives, debridase,collagenase, papain derivatives, streptokinase, sutilains, fibrinolysin,deoxyribonuclease, krill derivatives, trypsin or combinations thereof.Autolytic debridement relies on enhancing the natural process ofselective liquefaction, separation and digestion of necrotic tissue andeschar from healthy tissue that occurs in wounds due to macrophage andendogenous proteolytic activity. This is achieved by the use ofocclusive, semi-occlusive or moist interactive dressings. Enzymaticdebridement agents include a bromelain enriched enzyme product, othercollagenases, or other enzyme products capable of clearing devitalizedtissue or wound debris. NexoBrid™ (MediWound Ltd.) is one such bromelainenriched product that specifically targets heat-denatured collagen fordegradation, resulting in partial-thickness and full-thickness woundsrequiring a wound coverage or dressing product. Such products andmethods are described in U.S. Pat. Nos. 8,540,983; 8,119,124; 7,128,719;7,794,709; 8,624,077; and US2009/0010910A1, each of which isincorporated by reference herein.

In some aspects, the wound bed may include or be a chronic wound or anacute wound. Chronic wounds include but are not limited to venous legulcers, pressure ulcers, and diabetic foot ulcers. Acute wounds includebut are not limited to burns, traumatic injuries, amputation wounds,skin graft donor sites, bite wounds, frostbite wounds, dermabrasions,and surgical wounds.

In the cases where there is no dermis, biological products produced inaccordance with the present invention are utilized. The epidermis isremoved from such products (e.g., before dermis harvesting on the pigwith a VERSAJET™ Hydrosurgery system), so that just the dermis remains.Then, the subject biological product is placed on the patient'ssubcutaneous tissue, serving as a substrate for the cultured epidermalautograft process described herein.

In one aspect, a liver derived in accordance with the present disclosureis utilized for extracorporeal perfusion as a temporary filter for ahuman patient until a patient receives a human transplant. In anoperating area within the DPF Isolation Area, a source animal is placedunder a general anesthetic (ketamine, xylazine, enflurane) or euthanizedby captive bolt. A hepatectomy is then performed on the source animal indesignated pathogen free conditions. The liver product derived from thesource animal can be packaged and transported to the location of theprocedure in accordance with current practice with human donor livers.The procedure to utilize the liver filtration product can be performed,for example, by percutaneously cannulating a human patient's internaljugular vein for venous return with an arterial cannula andpercutaneously cannulating a patient's femoral vein for venous outflowwith an artery cannula. These cannulas are connected to a bypasscircuit, having a centrifugal pump, a heat exchanger, an oxygenator, anda roller pump incorporated therein. This circuit is primed withcrystalloids and run for a period of time (e.g., 10-30 minutes) beforethe liver from an animal according to the present disclosure isincorporated at a stabilized flow rate, e.g., 600-1000 ml/min,maintained in a crystalloid bath occasionally supplemented with warmsolution, e.g., 30-40° C.

It will be understood that, in the context of swine-to-humanxenotransplantation, each human recipient will have a majorhistocompatibility complex (MHC) (Class I, Class II and/or Class III)that is unique to that individual and will not match the MHC of thedonor swine. Accordingly, it will be understood that when a donor swinegraft is introduced to the recipient, the swine MHC molecules themselvesact as non-gal xeno-antigens, provoking an immune response from therecipient, leading to transplant rejection.

Human leukocyte antigen (HLA) genes show incredible sequence diversityin the human population. For example, there are >4,000 known alleles forthe HLA-B gene alone. The genetic diversity in HLA genes in whichdifferent alleles have different efficiencies for presenting differentantigens is believed to be a result of evolution conferring betterpopulation-level resistance against the wide range of differentpathogens to which humans are exposed. This genetic diversity alsopresents problems during xenotransplantation where the recipient'simmune response is the most important factor dictating the outcome ofengraftment and survival after transplantation.

In accordance with one aspect the present invention, a donor swine isprovided with a genome that is biologically engineered to express aspecific set of known human HLA molecules. Such HLA sequences areavailable, e.g., in the IPD-IMGT/HLA database (available atebi.ac.uk/ipd/imgt/hla/) and the international ImMunoGeneTicsInformation System® (available at imgt.org). For example, HLA-A1, B8,DR17 is the most common HLA haplotype among Caucasians, with a frequencyof 5%. Thus, the disclosed method can be performed using the knownMHC/HLA sequence information in combination with the disclosuresprovided herein.

In some aspects, the recipient's human leukocyte antigen (HLA) genes andMHC (Class I, II and/or III), are identified and mapped. It will beunderstood that ascertaining the human recipient's HLA/MHC sequence canbe done in any number of ways known in the art. For example, HLA/MHCgenes are usually typed with targeted sequencing methods: eitherlong-read sequencing or long-insert short-read sequencing.Conventionally, HLA types have been determined at 2-digit resolution(e.g., A*01), which approximates the serological antigen groupings. Morerecently, sequence specific oligonucleotide probes (SSOP) method hasbeen used for HLA typing at 4-digit resolution (e.g., A*01:01), whichcan distinguish amino acid differences. Currently, targeted DNAsequencing for HLA typing is the most popular approach for HLA typingover other conventional methods. Since the sequence-based approachdirectly determines both coding and non-coding regions, it can achieveHLA typing at 6-digit (e.g., A*01:01:01) and 8-digit (e.g.,A*01:01:01:01) resolution, respectively. HLA typing at the highestresolution is desirable to distinguish existing HLA alleles from newalleles or null alleles from clinical perspective. Such sequencingtechniques are described in, for example, Elsner H A, Blasczyk R: (2004)Immunogenetics of HLA null alleles: implications for blood stem celltransplantation. Tissue antigens. 64 (6): 687-695; Erlich R L, et al(2011) Next-generation sequencing for HLA typing of Class I loci. BMCgenomics. 12: 42-10.1186/1471-2164-12-42; Szolek A, et al. (2014)OptiType: Precision HLA typing from next-generation sequencing data.Bioinformatics 30:3310-3316; Nariai N, et al. (2015) HLA-VBSeq: AccurateHLA typing at full resolution from whole-genome sequencing data. BMCGenomics 16:S7; Dilthey A T, et al. (2016) High-accuracy HLA typeinference from whole-genome sequencing data using population referencegraphs. PLoS Comput Biol 12:e1005151; Xie C., et al. (2017) Fast andaccurate HLA typing from short-read next-generation sequence data withxHLA 114 (30) 8059-8064, each of which is incorporated herein in itsentirety by reference.

The known human HLA/MHC or an individual recipient's sequenced HLA/MHCsequence(s) may be utilized as a template to modify the swine leukocyteantigen (SLA)/MHC sequence to match, e.g., to have 80%, 85%, 90%, 95%,98%, 99%, or 100% sequence homology to a known human HLA/MHC sequence orthe human recipient's HLA/MHC sequence. Upon identifying a known humanrecipient HLA/MHC sequence to be used or performing genetic sequencingof a human recipient to obtain HLA/MHC sequences, biologicalreprogramming may be performed to SLA/MHC sequences in cells of theswine based on desired HLA/MHC sequences. For example, several targetingguide RNA (gRNA) sequences are administered to the swine of the presentdisclosure to reprogram SLA/MHC sequences in cells of the swine with thetemplate HLA/MHC sequences of the human recipient.

CRISPR-Cas9 is used to mediate rapid and scarless exchange of entire MHCalleles at specific native locus in swine cells. Multiplex targeting ofCas9 with two gRNAs is used to introduce single or double-strandedbreaks flanking the MHC allele, enabling replacement with the templateHLA/MHC sequence (provided as a single or double-stranded DNA template).In certain aspects, the CRISPR/Cas9 components are injected into swineoocytes, ova, zygotes, or blastocytes prior to transfer into fostermothers.

In certain aspects, the present disclosure includes embryogenesis andlive birth of SLA-free and HLA-expressing biologically reprogrammedswine. In certain aspects, the present disclosure includes breedingSLA-free and HLA-expressing biologically reprogrammed swine to createSLA-free and HLA-expressing progeny. In certain aspects, the CRISPR/Cas9components are injected into swine zygotes by intracytoplasmicmicroinjection of porcine zygotes. In certain aspects, the CRISPR/Cas9components are injected into swine prior to selective breeding of theCRISPR/Cas9 genetically modified swine. In certain aspects, theCRISPR/Cas9 components are injected into donor swine prior to harvestingcells, tissues, zygotes, and/or organs from the swine. In certainaspects, the CRISPR/Cas9 components include all necessary components forcontrolled gene editing including self-inactivation utilizing governinggRNA molecules as described in U.S. Pat. No. 9,834,791 (Zhang), which isincorporated herein by reference in its entirety.

The genetic modification can be made utilizing known genome editingtechniques, such as zinc-finger nucleases (ZFNs), transcriptionactivator-like effector nucleases (TALENs), adeno-associated virus(AAV)-mediated gene editing, and clustered regular interspacedpalindromic repeat Cas9 (CRISPR-Cas9). These programmable nucleasesenable the targeted generation of DNA double-stranded breaks (DSB),which promote the upregulation of cellular repair mechanisms, resultingin either the error-prone process of non-homologous end joining (NHEJ)or homology-directed repair (HDR), the latter of which can be used tointegrate exogenous donor DNA templates. CRISPR-Cas9 may also be used toremove viral infections in cells. For example, the genetic modificationvia CRISPR-Cas9 can be performed in a manner described in Kelton, W. et.al., “Reprogramming MHC specificity by CRISPR-Cas9-assisted cassetteexchange,” Nature, Scientific Reports, 7:45775 (2017) (“Kelton”), theentire disclosure of which is incorporated herein by reference.Accordingly, the present disclosure includes reprogramming usingCRISPR-Cas9 to mediate rapid and scarless exchange of entire alleles,e.g., MHC, HLA, SLA, etc.

In one aspect, the recipient's HLA/MHC gene is sequenced and templateHLA/MHC sequences are prepared based on the recipient's HLA/MHC genes.In another aspect, a known human HLA/MHC genotype from a WHO databasemay be used for genetic reprogramming of swine of the presentdisclosure. CRISPR-Cas9 plasmids are prepared, e.g., using polymerasechain reaction and the recipient's HLA/MHC sequences are cloned into theplasmids as templates. CRISPR cleavage sites at the SLA/MHC locus in theswine cells are identified and gRNA sequences targeting the cleavagesites and are cloned into one or more CRISPR-Cas9 plasmids. CRISPR-Cas9plasmids are then administered into the swine cells and CRIPSR/Cas9cleavage is performed at the MHC locus of the swine cells.

The SLA/MHC locus in the swine cells are replaced with one or moretemplate HLA/MHC sequences matching the known human HLA/MHC sequences orthe recipient's sequenced HLA/MHC genes. Cells of the swine aresequenced after performing the SLA/MHC reprogramming steps in order todetermine if the HLA/MHC sequences in the swine cells have beensuccessfully reprogrammed. One or more cells, tissues, and/or organsfrom the HLA/MHC sequence-reprogrammed swine are transplanted into ahuman recipient.

In certain aspects, HLA/MHC sequence-reprogrammed swine are bred for atleast one generation, or at least two generations, before their use as asource for live tissues, organs and/or cells used inxenotransplantation. In certain aspects, the CRISPR/Cas9 components canalso be utilized to inactivate genes responsible for PERV activity,e.g., the pol gene, thereby simultaneously completely eliminating PERVfrom the swine donors.

For purposes of modifying donor SLA/MHC to match recipient HLA/MHC,comparative genomic organization of the human and swinehistocompatibility complex has been mapped. For example, such SLA to HLAmapping can be found in: Lunney, J., “Molecular genetics of the swinemajor histocompatibility complex, the SLA complex,” Developmental andComparative Immunology 33: 362-374 (2009) (“Lunney”), the entiredisclosure of which is incorporated herein by reference. Accordingly, aperson of ordinary skill in the art effectively and efficientlygenetically reprogram swine cells in view of the present disclosure andusing the mapping of Lunney et al. as a reference tool.

The modification to the donor SLA/MHC to match recipient HLA/MHC causesexpression of specific MHC molecules from the swine cells that areidentical, or virtually identical, to the MHC molecules of a known humangenotype or the specific human recipient. In one aspect, the presentdisclosure involves making modifications limited to only specificportions of specific SLA regions of the swine's genome to retain aneffective immune profile in the swine while biological products arehypoimmunogenic when transplanted into human recipients such that use ofimmunosuppressants can be reduced or avoided. In contrast to aspects ofthe present disclosure, xenotransplantation studies of the prior artrequired immunosuppressant use to resist rejection. In one aspect, theswine genome is reprogrammed to knock-out swine genes corresponding toHLA-A, HLA-B, HLA-C, and DR, and to knock-in HLA-C, HLA-E, HLA-G. Insome aspects, the swine genome is reprogrammed to knock-out swine genescorresponding to HLA-A, HLA-B, HLA-C, HLA-F, DQ, and DR, and to knock-inHLA-C, HLA-E, HLA-G. In some aspects, the swine genome is reprogrammedto knock-out swine genes corresponding to HLA-A, HLA-B, HLA-C, HLA-F,DQ, and DR, and to knock-in HLA-C, HLA-E, HLA-G, HLA-F, and DQ. In oneaspect, the swine genome is reprogrammed to knock-out SLA-11; SLA-6,7,8;SLA-MIC2; and SLA-DQA; SLA-DQB1; SLA-DQB2, and to knock-in HLA-C; HLA-E;HLA-G; and HLA-DQ. In certain aspects, HLA-C expression is reduced inthe reprogrammed swine genome. By reprogramming the swine cells to beinvisible to a human's immune system, this reprogramming therebyminimizes or even eliminates an immune response that would haveotherwise occurred based on swine MHC molecules otherwise expressed fromthe donor swine cells.

It will therefore be understood that this aspect (i.e., reprogrammingthe SLA/MHC to express specifically selected human MHC alleles), whenapplied to swine cells, tissues, and organs for purposes ofxenotransplantation will decrease rejection as compared to cells,tissues, and organs derived from a wild-type swine or otherwisegenetically modified swine that lacks this reprogramming, e.g.,transgenic swine or swine with non-specific or different geneticmodifications.

It will be further understood that causing the donor swine cells,tissues, and organs to express a known human MHC genotype or therecipient's MHC specifically as described herein, combined with theelimination in the donor swine cells of alpha-1,3-galactosytransferase,Neu5Gc, and β1,4-N-acetylgalactosaminyltransferase (B4GALNT2) (e.g.,“single knockout,” “double knockout,” or “triple knockout”), presents aswine whose cells will have a decreased immunological rejection ascompared to a triple knockout swine that lacks the specific SLA/MHCreprogramming of the present disclosure.

Cryopreservation and storage according to the present disclosureincludes preparing biological product according to the presentdisclosures, placing in a container, adding freeze media to thecontainer and sealing. For example. 15% dimethyl sulfoxide (DMSO)cryoprotective media is combined with fetal porcine serum (FPS) or donorserum (if FPS is unavailable) in a 1:1 ratio, filtered (0.45 micron),and chilled to 4° C. prior to use. The containers are subsequentlyfrozen in a controlled rate, phase freezer at a rate of 1° C. per minuteto −40° C., then rapidly cooled to a temperature −80° C. DMSO displacesintracellular fluid during the freezing process. Cryoprotective media,e.g., CryoStor is used in an amount of about 40-80%, or 50-70% based onmaximum internal volume of the cryovial (10 ml) less the volume of thexenotransplantation product. In order to thaw the cryopreservedbiological product for surgical use, sealed vials were placed in ˜37° C.water baths for approximately 0.5 to 2 minutes, at which point thecontainer is opened and the product was removed using sterile technique.Subsequently, products undergo three, 1-minute serial washes, e.g., insaline with gentle agitation, in order to dilute and systematicallyremove ambient, residual DMSO and prevent loss of cell viability. Theproduct may then be used surgically.

It will be understood that the xenotransplantation product may beprocessed, stored, transported, and/or otherwise handled usingmaterials, containers and processes to ensure preserved sterility andprevent damage thereto. In some aspects, a sterile non-adhesive materialmay be used to protect the xenotransplantation product, e.g., to supportthe xenotransplantation product and prevent adhesive of the product tosurfaces and/or to prevent self-adhesion of the xenotransplantationproduct during manipulation, storage, or transport. Unintentionaladhesion of the xenotransplantation product may disrupt the integrity ofthe xenotransplantation product and potentially reduce its therapeuticviability. Inclusion of the sterile non-adhesive material providesprotection and/or physical support and prevents adhesion. In someaspects, the sterile non-adhesive material is not biologically orchemically active and does not directly impact the metabolic activity orefficacy of the xenotransplantation product itself.

Aspects of the present disclosure are further described by the followingnon-limiting list of items:

Item 1. A biological system for generating and preserving a repositoryof personalized, humanized transplantable cells, tissues, and organs fortransplantation, wherein the biological system is biologically activeand metabolically active, the biological system comprising geneticallyreprogrammed cells, tissues, and organs in a non-human animal fortransplantation into a human recipient,

wherein the non-human animal is a genetically reprogrammed swine forxenotransplantation of cells, tissue, and/or an organ isolated from thegenetically reprogrammed swine, the genetically reprogrammed swinecomprising a nuclear genome that has been reprogrammed to replace aplurality of nucleotides in a plurality of exon regions of a majorhistocompatibility complex of a wild-type swine with a plurality ofsynthesized nucleotides from a human captured reference sequence, and

wherein cells of said genetically reprogrammed swine do not present oneor more surface glycan epitopes selected from alpha-Gal, Neu5Gc, andSD^(a),

and

wherein genes encoding alpha-1,3 galactosyltransferase, cytidinemonophosphate-N-acetylneuraminic acid hydroxylase (CMAH), andβ1,4-N-acetylgalactosaminyltransferase are altered such that thegenetically reprogrammed swine lacks functional expression of surfaceglycan epitopes encoded by said genes,

wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of: i) at least one of thewild-type swine's SLA-1, SLA-2, and SLA-3 with nucleotides from anorthologous exon region of HLA-A, HLA-B, and HLA-C, respectively, of thehuman captured reference sequence; and ii) at least one the wild-typeswine's SLA-6, SLA-7, and SLA-8 with nucleotides from an orthologousexon region of HLA-E, HLA-F, and HLA-G, respectively, of the humancaptured reference sequence; and iii) at least one of the wild-typeswine's SLA-DR and SLA-DQ with nucleotides from an orthologous exonregion of HLA-DR and HLA-DQ, respectively, of the human capturedreference sequence,

wherein the reprogrammed genome comprises at least one of A-C:

A) wherein the reprogrammed swine nuclear genome comprises site-directedmutagenic substitutions of nucleotides at exon regions of the wild-typeswine's β2-microglobulin with nucleotides from orthologous exons of aknown human β2-microglobulin from the human captured reference sequence;B) wherein the reprogrammed swine nuclear genome comprises apolynucleotide that encodes a polypeptide that is a humanized beta 2microglobulin (hB2M) polypeptide sequence that is at least 95% identicalto the amino acid sequence of beta 2 microglobulin glycoproteinexpressed by the human captured reference genome;C) wherein the reprogrammed swine nuclear genome has been reprogrammedsuch that, at the swine's endogenous β2-microglobulin locus, the nucleargenome has been reprogrammed to comprise a nucleotide sequence encodingβ2-microglobulin polypeptide of the human recipient,

wherein the reprogrammed swine nuclear genome has been reprogrammed suchthat the genetically reprogrammed swine lacks functional expression ofthe wild-type swine's endogenous β2-microglobulin polypeptides, and

wherein said reprogramming does not introduce any frameshifts or framedisruptions.

Item 2. The biological system of item 1, wherein the geneticallyreprogrammed swine is non-transgenic.

Item 3. The biological system of item 1 or item 2, wherein intronregions of the wild-type swine's genome are not reprogrammed.

Item 4. The biological system of any one of or combination of items 1-3,wherein said genetically reprogrammed swine is free of at least thefollowing pathogens: Ascaris species, Cryptosporidium species,Echinococcus, Strongyloids sterocolis, Toxoplasma gondii, Brucella suis,Leptospira species, Mycoplasma hyopneumoniae, porcine reproductive andrespiratory syndrome, pseudorabies, Staphylococcus species, Microphytonspecies, Trichophyton species, porcine influenza, porcinecytomegalovirus, arterivirus, coronavirus, Bordetella bronchiseptica,and Livestock-associated methicillin-resistant Staphylococcus aureus.

Item 5. The biological system of any one of or combination of items 1-4,wherein said genetically reprogrammed swine is maintained according to abioburden-reducing procedure, said procedure comprising maintaining theswine in an isolated closed herd, wherein all other animals in theisolated closed herd are confirmed to be free of said pathogens, andwherein the swine is isolated from contact with any non-human animalsand animal housing facilities outside of the isolated closed herd.

Item 6. The biological system of any one of or combination of items 1-4,wherein the wild-type swine genome comprises reprogrammed nucleotides atSLA-MIC-2 gene and at exon regions encoding SLA-3, SLA-6, SLA-7, SLA-8,SLA-DQ, CTLA-4, PD-L1, EPCR, TBM, TFPI, and beta-2-microglobulin usingthe human capture reference sequence, wherein the human cell, tissue, ororgan lacks functional expression of swine beta-2-microglobulin, SLA-1,SLA-2, and SLA-DR.

Item 7. The biological system of any one of or combination of items 1-5,wherein the wild-type swine genome comprises reprogrammed nucleotides atone or more of a CTLA-4 promoter and a PD-L1 promoter, wherein the oneor more of the CTLA-4 promoter and the PD-L1 promoter are reprogrammedto increase expression of one or both of reprogrammed CTLA-4 andreprogrammed PD-L1 compared to the wild-type swine's endogenousexpression of CTLA-4 and PD-L1.

Item 8. The biological system of any one of or combination of items 1-6,wherein a total number of the synthesized nucleotides is equal to atotal number of the replaced nucleotides, such that there is no net lossor net gain in number of nucleotides after reprogramming the genome ofthe wild-type swine with the synthesized nucleotides.

Item 9. The biological system of any one of or combination of items 1-7,wherein the reprogramming with the plurality of synthesized nucleotidesdo not include replacement of nucleotides in codon regions that encodeamino acids that are conserved between the wild-type swine MHC sequenceand the human captured reference sequence

Item 10. The biological system of any one of or combination of items1-8, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at the major histocompatibility complex ofthe wild-type swine with orthologous nucleotides from the human capturedreference sequence.

Item 11. The biological system of any one of or combination of items1-9, wherein site-directed mutagenic substitutions are made in germ-linecells used to produce the non-human animal.

Item 12. The biological system of any one of or combination of items1-10, wherein the human captured reference sequence is a human patientcapture sequence, a human population-specific human capture sequence, oran allele-group-specific human capture sequence.

Item 13. The biological system of any one of or combination of items1-11, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type swine'sSLA-1 with nucleotides from an orthologous exon region of a HLA-Acaptured reference sequence.

Item 14. The biological system of any one of or combination of items1-12, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type swine'sSLA-2 with nucleotides from an orthologous exon region of a HLA-Bcaptured reference sequence.

Item 14. The biological system of any one of or combination of items1-13, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type swine'sSLA-3 with nucleotides from an orthologous exon region of a HLA-Ccaptured reference sequence.

Item 15. The biological system of any one of or combination of items1-14, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type swine'sSLA-6 with nucleotides from an orthologous exon region of a HLA-Ecaptured reference sequence.

Item 16. The biological system of any one of or combination of items1-15, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type swine'sSLA-7 with nucleotides from an orthologous exon region of a HLA-Fcaptured reference sequence.

Item 17. The biological system of any one of or combination of items1-16, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type swine'sSLA-8 with nucleotides from an orthologous exon region of a HLA-Gcaptured reference sequence.

Item 18. The biological system of any one of or combination of items1-17, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type swine'sMHC class I chain-related 2 (MIC-2).

Item 19. The biological system of any one of or combination of items1-18, wherein the reprogrammed genome lacks functional expression ofSLA-1, SLA-2, SLA-DR, or a combination thereof.

Item 20. The biological system of any one of or combination of items1-19, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type swine'sSLA-DQA from an orthologous exon region of a HLA-DQA1 captured referencesequence.

Item 21. The biological system of any one of or combination of items1-20, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type swine'sSLA-DQB from an orthologous exon region of a HLA-DQB1 captured referencesequence.

Item 22. The biological system of any one of or combination of items1-21, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type swine'sSLA-DRA and SLA-DRB1 with nucleotides from orthologous exon regions ofHLA-DRA1 and HLA-DRB1 of the human captured reference sequence, orwherein the reprogrammed genome lacks functional expression of SLA-DRAand SLA-DRB1.

Item 23. The biological system of any one of or combination of items1-22, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type swine'sSLA-DQA and SLA-DQB1 with nucleotides from orthologous exon regions ofHLA-DQA1 and HLA-DQB1 of the human captured reference sequence.

Item 24. The biological system of any one of or combination of items1-23, wherein the site-directed mutagenic substitutions of nucleotidesare at codons that are not conserved between the wild-type swine'snuclear genome and the known human sequence.

Item 25. The biological system of any one of or combination of items1-24, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type swine'sB2-microglobulin with nucleotides from orthologous exons of a knownhuman B2-microglobulin.

Item 26. The biological system of any one of or combination of items1-25, wherein the reprogrammed swine nuclear genome comprises apolynucleotide that encodes a polypeptide that is a humanized beta 2microglobulin (hB2M) polypeptide sequence that is at least 95% identicalto the amino acid sequence of beta 2 microglobulin glycoproteinexpressed by the human captured reference genome;

Item 27. The biological system of any one of or combination of items1-26, wherein said nuclear genome has been reprogrammed such that thegenetically reprogrammed swine lacks functional expression of thewild-type swine's endogenous β2-microglobulin polypeptides.

Item 28. The biological system of any one of or combination of items1-27, wherein said nuclear genome has been reprogrammed such that, atthe swine's endogenous β2-microglobulin locus, the nuclear genome hasbeen reprogrammed to comprise a nucleotide sequence encodingβ2-microglobulin polypeptide of the human captured reference sequence.

Item 29. The biological system of any one of or combination of items1-28, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of SLA-3, SLA-6, SLA-7,SLA-8, and MIC-2.

Item 30. The biological system of any one of or combination of items1-29, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of SLA-DQ and MIC-2.

Item 31. The biological system of any one of or combination of items1-30, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at SLA-3, SLA-6, SLA-7, SLA-8, SLA-DQ, andMIC-2.

Item 32. The biological system of any one of or combination of items1-31, wherein the reprogrammed genome lacks functional expression ofSLA-DR, SLA-1, and/or SLA-2.

Item 33. The biological system of any one of or combination of items1-32, wherein the nuclear genome is reprogrammed using scarless exchangeof the exon regions, wherein there are no frameshifts, insertionmutations, deletion mutations, missense mutations, and nonsensemutations.

Item 34. The biological system of any one of or combination of items1-33, wherein the nuclear genome is reprogrammed without introduction ofany net insertions, deletions, truncations, or other genetic alterationsthat would cause a disruption of protein expression via frame shift,nonsense, or missense mutations.

Item 35. The biological system of any one of or combination of items1-34, wherein nucleotides in intron regions of the nuclear genome arenot altered.

Item 36. The biological system of any one of or combination of items1-35, wherein said nuclear genome is reprogrammed to be homozygous atthe reprogrammed exon regions.

Item 37. The biological system of any one of or combination of items1-36, wherein said nuclear genome is reprogrammed such thatextracellular, phenotypic surface expression of polypeptide istolerogenic in a human recipient.

Item 38. The biological system of any one of or combination of items1-37, wherein said nuclear genome is reprogrammed such that expressionof cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is increased byreprogramming a CTLA-4 promoter sequence.

Item 39. The biological system of any one of or combination of items1-38, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type CTLA-4with nucleotides from orthologous exons of a human captured referencesequence CTLA-4.

Item 40. The biological system of any one of or combination of items1-39, wherein the reprogrammed nuclear genome comprises a polynucleotidethat encodes a protein that is a humanized CTLA-4 polypeptide sequencethat is at least 95% identical to CTLA-4 expressed by the human capturedreference genome.

Item 41. The biological system of any one of or combination of items1-40, wherein said nuclear genome is reprogrammed such that expressionof Programmed death-ligand 1 (PD-L1) is increased by reprogramming aPD-L1 promoter sequence.

Item 42. The biological system of any one of or combination of items1-41, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type PD-L1 withnucleotides from orthologous exons of a known human PD-L1.

Item 43. The biological system of any one of or combination of items1-42, wherein the reprogrammed nuclear genome comprises a polynucleotidethat encodes a protein that is a humanized PD-L1 polypeptide sequencethat is at least 95% identical to PD-L1 expressed by the human capturedreference genome.

Item 44. A genetically reprogrammed, biologically active andmetabolically active non-human cell, tissue, or organ obtained from thebiological system of any one of or combination of items 1-43.

Item 45. The genetically reprogrammed, biologically active andmetabolically active non-human cell, tissue, or organ of item 44,wherein the genetically reprogrammed, biologically active andmetabolically active non-human cell is a stem cell, an embryonic stemcell, a pluripotent stem cell, or a differentiated stem cell.

Item 46. The genetically reprogrammed, biologically active andmetabolically active non-human cell, tissue, or organ of item 45,wherein the stem cell is a hematopoietic stem cell.

Item 47. The genetically reprogrammed, biologically active andmetabolically active non-human cell, tissue, or organ of item 44,wherein the genetically reprogrammed, biologically active andmetabolically active non-human tissue is a nerve, cartilage, or skin.

Item 48. The genetically reprogrammed, biologically active andmetabolically active non-human cell, tissue, or organ of item 44,wherein the genetically reprogrammed, biologically active andmetabolically active non-human organ is a solid organ.

Item 49. A method of preparing a genetically reprogrammed swinecomprising a nuclear genome that lacks functional expression of surfaceglycan epitopes selected from alpha-Gal, Neu5Gc, and SD^(a) and isgenetically reprogrammed to express a humanized phenotype of a humancaptured reference sequence comprising:

-   -   a. obtaining a porcine fetal fibroblast cell, a porcine zygote,        a porcine Induced Pluripotent Stem Cells (IPSC), or a porcine        germ-line cell;    -   b. genetically altering said cell in a) to lack functional        alpha-1,3 galactosyltransferase, cytidine        monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and        β1,4-N-acetylgalactosaminyltransferase;    -   c. genetically reprogramming said cell in b) using clustered        regularly interspaced short palindromic repeats (CRISPR)/Cas for        site-directed mutagenic substitutions of nucleotides at exon        regions of: i) at least one of the wild-type swine's SLA-1,        SLA-2, and SLA-3 with nucleotides from an orthologous exon        region of HLA-A, HLA-B, and HLA-C, respectively, of the human        captured reference sequence; and ii) at least one the wild-type        swine's SLA-6, SLA-7, and SLA-8 with nucleotides from an        orthologous exon region of HLA-E, HLA-F, and HLA-G,        respectively, of the human captured reference sequence; and iii)        at least one of the wild-type swine's SLA-DR and SLA-DQ with        nucleotides from an orthologous exon region of HLA-DR and        HLA-DQ, respectively, of the human captured reference sequence,        wherein intron regions of the wild-type swine's genome are not        reprogrammed, and

wherein the reprogrammed genome comprises at least one of A-C:

A) wherein the reprogrammed swine nuclear genome comprises site-directedmutagenic substitutions of nucleotides at exon regions of the wild-typeswine's β2-microglobulin with nucleotides from orthologous exons of aknown human β2-microglobulin from the human captured reference sequence;B) wherein the reprogrammed swine nuclear genome comprises apolynucleotide that encodes a polypeptide that is a humanized beta 2microglobulin (hB2M) polypeptide sequence that is at least 95% identicalto beta 2 microglobulin expressed by the human captured referencegenome;C) wherein the reprogrammed swine nuclear genome has been reprogrammedsuch that the genetically reprogrammed swine lacks functional expressionof the wild-type swine's endogenous β2-microglobulin polypeptides,wherein the reprogrammed swine nuclear genome has been reprogrammed suchthat, at the swine's endogenous β2-microglobulin locus, the nucleargenome has been reprogrammed to comprise a nucleotide sequence encodingβ2-microglobulin polypeptide of the human recipient,

wherein said reprogramming does not introduce any frameshifts or framedisruptions,

-   -   d. generating an embryo from the genetically reprogrammed cell        in c); and    -   e. transferring the embryo into a surrogate pig and growing the        transferred embryo in the surrogate pig.

Item 50. The method of item 49, wherein step (a) further comprisesreplacing a plurality of nucleotides in a plurality of exon regions of amajor histocompatibility complex of a wild-type swine with nucleotidesfrom orthologous exon regions of a major histocompatibility complexsequence from the human captured reference sequence, wherein saidreplacing does not introduce any frameshifts or frame disruptions.

Item 51. The method of any one of or combination of items 49-50, whereinsaid replacing comprises performing site-directed mutagenicsubstitutions of nucleotides at the major histocompatibility complex ofthe wild-type swine with orthologous nucleotides from the known humanmajor histocompatibility complex sequence.

Item 52. The method of any one of or combination of items 49-51, whereinthe human captured reference sequence is a human patient capturesequence, a human population-specific human capture sequence, or anallele-group-specific human capture sequence.

Item 53. The method of any one of or combination of items 49-52, whereinthe orthologous exon regions are at one or more polymorphicglycoproteins of the wild-type swine's major histocompatibility complex.

Item 54. The method of any one of or combination of items 49-53, furthercomprising: impregnating the surrogate pig with the embryo, gestatingthe embryo, and delivering a piglet from the surrogate pig throughCesarean section,

confirming that said piglet is free of at least the following zoonoticpathogens:

(i) Ascaris species, Cryptosporidium species, Echinococcus, Strongyloidssterocolis, and Toxoplasma gondii in fecal matter;

(ii) Leptospira species, Mycoplasma hyopneumoniae, porcine reproductiveand respiratory syndrome virus (PRRSV), pseudorabies, transmissiblegastroenteritis virus (TGE)/Porcine Respiratory Coronavirus, andToxoplasma Gondii by determining antibody titers;

(iii) Porcine Influenza;

(iv) the following bacterial pathogens as determined by bacterialculture: Bordetella bronchisceptica, Coagulase-positive staphylococci,Coagulase-negative staphylococci, Livestock-associated methicillinresistant Staphylococcus aureus (LA MRSA), Microphyton and Trichophytonspp.;(v) Porcine cytomegalovirus; and(vi) Brucella suis; andmaintaining the piglet according to a bioburden-reducing procedure, saidprocedure comprising maintaining the piglet in an isolated closed herd,wherein all other animals in the isolated closed herd are confirmed tobe free of said zoonotic pathogens, wherein the piglet is isolated fromcontact with any non-human animals and animal housing facilities outsideof the isolated closed herd.

Item 55. The method of any one of or combination of items 49-54, whereinthe wild-type swine genome comprises reprogrammed nucleotides atSLA-MIC-2 gene and at exon regions encoding SLA-3, SLA-6, SLA-7, SLA-8,SLA-DQ, CTLA-4, PD-L1, EPCR, TBM, TFPI, and beta-2-microglobulin usingthe human capture reference sequence, wherein the human cell, tissue, ororgan lacks functional expression of swine beta-2-microglobulin, SLA-DR,SLA-1, and SLA-2.

Item 56. The method of any one of or combination of items 49-55, whereinthe wild-type swine genome comprises reprogrammed nucleotides at one ormore of a CTLA-4 promoter and a PD-L1 promoter, wherein the one or moreof the CTLA-4 promoter and the PD-L1 promoter are reprogrammed toincrease expression of one or both of reprogrammed CTLA-4 andreprogrammed PD-L1 compared to the wild-type swine's endogenousexpression of CTLA-4 and PD-L1.

Item 57. The method of any one of or combination of items 49-56, whereina total number of the synthesized nucleotides is equal to a total numberof the replaced nucleotides, such that there is no net loss or net gainin number of nucleotides after reprogramming the genome of the wild-typeswine with the synthesized nucleotides.

Item 58. The method of any one of or combination of items 49-57, whereinthe reprogramming with the plurality of synthesized nucleotides do notinclude replacement of nucleotides in codon regions that encode aminoacids that are conserved between the wild-type swine MHC sequence andthe human captured reference sequence

Item 59. The method of any one of or combination of items 49-58, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at the major histocompatibility complex of the wild-typeswine with orthologous nucleotides from the human captured referencesequence.

Item 60. The method of any one of or combination of items 49-59, whereinsite-directed mutagenic substitutions are made in germ-line cells usedto produce the non-human animal.

Item 61. The method of any one of or combination of items 49-60, whereinthe human captured reference sequence is a human patient capturesequence, a human population-specific human capture sequence, or anallele-group-specific human capture sequence.

Item 62. The method of any one of or combination of items 49-61, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of the wild-type swine's SLA-1 withnucleotides from an orthologous exon region of a HLA-A capturedreference sequence.

Item 63. The method of any one of or combination of items 49-62, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of the wild-type swine's SLA-2 withnucleotides from an orthologous exon region of a HLA-B capturedreference sequence.

Item 64. The method of any one of or combination of items 49-63, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of the wild-type swine's SLA-3 withnucleotides from an orthologous exon region of a HLA-C capturedreference sequence.

Item 65. The method of any one of or combination of items 49-64, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of the wild-type swine's SLA-6 withnucleotides from an orthologous exon region of a HLA-E capturedreference sequence.

Item 66. The method of any one of or combination of items 49-65, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of the wild-type swine's SLA-7 withnucleotides from an orthologous exon region of a HLA-F capturedreference sequence.

Item 67. The method of any one of or combination of items 49-66, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of the wild-type swine's SLA-8 withnucleotides from an orthologous exon region of a HLA-G capturedreference sequence.

Item 68. The method of any one of or combination of items 49-67, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of the wild-type swine's MHC class Ichain-related 2 (MIC-2).

Item 69. The method of any one of or combination of items 49-68, whereinthe reprogrammed genome lacks functional expression of SLA-1, SLA-2,SLA-DR, or a combination thereof.

Item 70. The method of any one of or combination of items 49-69, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of the wild-type swine's SLA-DQA from anorthologous exon region of a HLA-DQA1 captured reference sequence.

Item 71. The method of any one of or combination of items 49-70, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of the wild-type swine's SLA-DQB from anorthologous exon region of a HLA-DQB1 captured reference sequence.

Item 72. The method of any one of or combination of items 49-71, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of the wild-type swine's SLA-DRA andSLA-DRB1 with nucleotides from orthologous exon regions of HLA-DRA1 andHLA-DRB1 of the human captured reference sequence.

Item 73. The method of any one of or combination of items 49-72, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of the wild-type swine's SLA-DQA andSLA-DQB1 with nucleotides from orthologous exon regions of HLA-DQA1 andHLA-DQB1 of the human captured reference sequence.

Item 74. The method of any one of or combination of items 49-73, whereinthe site-directed mutagenic substitutions of nucleotides are at codonsthat are not conserved between the wild-type swine's nuclear genome andthe known human sequence.

Item 75. The method of any one of or combination of items 49-74, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of the wild-type swine's B2-microglobulinwith nucleotides from orthologous exons of a known humanB2-microglobulin.

Item 76. The method of any one of or combination of items 49-75, whereinthe reprogrammed swine nuclear genome comprises a polynucleotide thatencodes a polypeptide that is a humanized beta 2 microglobulin (hB2M)polypeptide sequence that is at least 95% identical to the amino acidsequence of beta 2 microglobulin glycoprotein expressed by the humancaptured reference genome;

Item 77. The method of any one of or combination of items 49-76, whereinsaid nuclear genome has been reprogrammed such that the geneticallyreprogrammed swine lacks functional expression of the wild-type swine'sendogenous β2-microglobulin polypeptides.

Item 78. The method of any one of or combination of items 49-77, whereinsaid nuclear genome has been reprogrammed such that, at the swine'sendogenous β2-microglobulin locus, the nuclear genome has beenreprogrammed to comprise a nucleotide sequence encoding β2-microglobulinpolypeptide of the human captured reference sequence.

Item 79. The method of any one of or combination of items 49-78, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of SLA-3, SLA-6, SLA-7, SLA-8, and MIC-2.

Item 80. The method of any one of or combination of items 49-79, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of SLA-DQ, and MIC-2.

Item 81. The method of any one of or combination of items 49-80, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at SLA-3, SLA-6, SLA-7, SLA-8, SLA-DQ, and MIC-2.

Item 82. The method of any one of or combination of items 49-81, whereinthe reprogrammed genome lacks functional expression of SLA-DR, SLA-1,and/or SLA-2.

Item 83. The method of any one of or combination of items 49-82, whereinthe nuclear genome is reprogrammed using scarless exchange of the exonregions, wherein there are no frameshifts, insertion mutations, deletionmutations, missense mutations, and nonsense mutations.

Item 84. The method of any one of or combination of items 49-83, whereinthe nuclear genome is reprogrammed without introduction of any netinsertions, deletions, truncations, or other genetic alterations thatwould cause a disruption of protein expression via frame shift,nonsense, or missense mutations.

Item 85. The method of any one of or combination of items 49-84, whereinnucleotides in intron regions of the nuclear genome are not altered.

Item 86. The method of any one of or combination of items 49-85, whereinsaid nuclear genome is reprogrammed to be homozygous at the reprogrammedexon regions.

Item 87. The method of any one of or combination of items 49-86, whereinsaid nuclear genome is reprogrammed such that extracellular, phenotypicsurface expression of polypeptide is tolerogenic in a human recipient.

Item 88. The method of any one of or combination of items 49-87, whereinsaid nuclear genome is reprogrammed such that expression of cytotoxicT-lymphocyte-associated protein 4 (CTLA-4) is increased by reprogramminga CTLA-4 promoter sequence.

Item 89. The method of any one of or combination of items 49-88, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of the wild-type CTLA-4 with nucleotidesfrom orthologous exons of a human captured reference sequence CTLA-4.

Item 90. The method of any one of or combination of items 49-89, whereinthe reprogrammed nuclear genome comprises a polynucleotide that encodesa protein that is a humanized CTLA-4 polypeptide sequence that is atleast 95% identical to CTLA-4 expressed by the human captured referencegenome.

Item 91. The method of any one of or combination of items 49-90, whereinsaid nuclear genome is reprogrammed such that expression of Programmeddeath-ligand 1 (PD-L1) is increased by reprogramming a PD-L1 promotersequence.

Item 92. The method of any one of or combination of items 49-91, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of the wild-type PD-L1 with nucleotidesfrom orthologous exons of a known human PD-L1.

Item 93. The method of any one of or combination of items 49-92, whereinthe reprogrammed nuclear genome comprises a polynucleotide that encodesa protein that is a humanized PD-L1 polypeptide sequence that is atleast 95% identical to PD-L1 expressed by the human captured referencegenome.

Item 94. A method of inducing at least partial immunological tolerancein a recipient human to a xenotransplanted cell, tissue, or organ, themethod comprising:

producing or obtaining non-human cell, tissue, or organ obtained fromthe biological system of any one of or combination of items 1-48,wherein the wild-type swine genome comprises reprogrammed nucleotides atSLA-MIC-2 gene and at exon regions of one or more encoding the wild-typeswine's MHC Class Ia, MHC class Ib, MHC Class II, andbeta-2-microglobulin using the human capture reference sequence andwherein the human cell, tissue, or organ lacks functional expression ofswine beta-2-microglobulin; andimplanting the non-human cell, tissue, or organ into the recipienthuman.

Item 95. A method of reducing Natural Killer cell-mediated rejection ofa xenograft comprising: producing or obtaining non-human cell, tissue,or organ obtained from the biological system of any one of orcombination of items 1-48, wherein the wild-type swine genome comprisesreprogrammed nucleotides at SLA-MIC-2 gene and at exon regions encodingone or more of the wild-type swine's MHC Class Ia, MHC class Ib, MHCClass II, and beta-2-microglobulin using the human capture referencesequence and wherein the human cell, tissue, or organ lacks functionalexpression of swine beta-2-microglobulin, and wherein the wild-typeswine genome comprises reprogrammed nucleotides at exon regions encodingone or more of the wild-type swine's CTLA-4 and PD-L1; and implantingthe non-human cell, tissue, or organ into the recipient human.

Item 96. A method of reducing Cytotoxic T-cell Lymphocyte cell-mediatedrejection of a xenograft comprising:

producing or obtaining non-human cell, tissue, or organ obtained fromthe biological system of any one of or combination of items 1-48,wherein the wild-type swine genome comprises reprogrammed nucleotides atSLA-MIC-2 gene and at exon regions encoding one or more of the wild-typeswine's MHC Class Ia, MHC class Ib, MHC Class II, andbeta-2-microglobulin using the human capture reference sequence andwherein the human cell, tissue, or organ lacks functional expression ofswine beta-2-microglobulin, and wherein the wild-type swine genomecomprises reprogrammed nucleotides at exon regions encoding one or moreof the wild-type swine's CTLA-4 and PD-L1; and implanting the non-humancell, tissue, or organ into the recipient human.

Item 97. A method of preventing or reducing coagulation and/orthrombotic ischemia in a recipient human to a xenotransplanted cell,tissue, or organ, the method comprising:

producing or obtaining non-human cell, tissue, or organ obtained fromthe biological system of any one of or combination of items 1-48,wherein the wild-type swine genome comprises reprogrammed nucleotides atSLA-MIC-2 gene and at exon regions encoding one or more of the wild-typeswine's MHC Class Ia, MHC class Ib, MHC Class II, andbeta-2-microglobulin using the human capture reference sequence, whereinthe human cell, tissue, or organ lacks functional expression of swinebeta-2-microglobulin, and wherein the wild-type swine genome comprisesreprogrammed nucleotides at exon regions encoding one or more of thewild-type swine's endothelial protein C receptor (EPCR), thrombomodulin(TBM), and tissue factor pathway inhibitor (TFPI); andimplanting the non-human cell, tissue, or organ into the recipienthuman.

Item 98. A method of reducing MHC Class Ia-mediated rejection of axenograft comprising:

producing or obtaining non-human cell, tissue, or organ obtained fromthe biological system of any one of or combination of items 1-48,wherein the wild-type swine genome comprises reprogrammed nucleotides atSLA-MIC-2 gene and at exon regions encoding SLA-3 and one or more of thewild-type swine's MHC class Ib, MHC Class II, and beta-2-microglobulinusing the human capture reference sequence, wherein the human cell,tissue, or organ lacks functional expression of swinebeta-2-microglobulin, SLA-1, and SLA-2; and implanting the non-humancell, tissue, or organ into the recipient human.

Item 99. A method of reducing MHC Class Ib-mediated rejection of axenograft comprising:

producing or obtaining non-human cell, tissue, or organ obtained fromthe biological system of any one of or combination of items 1-48,wherein the wild-type swine genome comprises reprogrammed nucleotides atSLA-MIC-2 gene and at exon regions encoding SLA-6, SLA-7, and SLA-8, andone or more of the wild-type swine's MHC class Ia, MHC Class II, andbeta-2-microglobulin using the human capture reference sequence, whereinthe human cell, tissue, or organ lacks functional expression of swinebeta-2-microglobulin; and implanting the non-human cell, tissue, ororgan into the recipient human.

Item 100. A method of reducing MHC Class II-mediated rejection of axenograft comprising:

producing or obtaining non-human cell, tissue, or organ obtained fromthe biological system of any one of or combination of items 1-48,wherein the wild-type swine genome comprises reprogrammed nucleotides atSLA-MIC-2 gene and at exon regions encoding at least one of SLA-DR andSLA-DQ, and one or more of the wild-type swine's MHC class Ia, MHC ClassIb, and beta-2-microglobulin using the human capture reference sequence,wherein the human cell, tissue, or organ lacks functional expression ofswine beta-2-microglobulin; and implanting the non-human cell, tissue,or organ into the recipient human.

Item 101. A method of inhibiting apoptotic cell-mediated rejection of axenograft comprising:

producing or obtaining non-human cell, tissue, or organ obtained fromthe biological system of any one of or combination of items 1-48,wherein the wild-type swine genome comprises reprogrammed nucleotides atSLA-MIC-2 gene and at exon regions encoding one or more of the wild-typeswine's MHC Class Ia, MHC class Ib, MHC Class II, andbeta-2-microglobulin using the human capture reference sequence andwherein the human cell, tissue, or organ lacks functional expression ofswine beta-2-microglobulin, and wherein the wild-type swine genomecomprises reprogrammed nucleotides at exon regions encoding one or moreof the wild-type swine's CTLA-4 and PD-L1; and

implanting the non-human cell, tissue, or organ into the recipienthuman.

Item 102. A method of producing a donor swine tissue or organ forxenotransplantation, wherein cells of said donor swine tissue or organare genetically reprogrammed to be characterized by a recipient-specificsurface phenotype comprising:

obtaining a biological sample containing DNA from a prospective humantransplant recipient; performing whole genome sequencing of thebiological sample to obtain a human capture reference sequence;

comparing the human capture reference sequence with the wild-type genomeof the donor swine at loci (i)-(v):

(i) exon regions encoding at least one of SLA-1, SLA-2, and SLA-3;

(ii) exon regions encoding at least one of SLA-6, SLA-7, and SLA-8;

(iii) exon regions encoding at least one of SLA-DR and SLA-DQ;

(iv) one or more exons encoding beta 2 microglobulin (B2M);

(v) exon regions of SLA-MIC-2 gene and a gene encoding at least one ofPD-L1, CTLA-4, EPCR, TBM, and TFPI,

creating synthetic donor swine nucleotide sequences of 10 to 350basepairs in length for one or more of said loci (i)-(v), wherein saidsynthetic donor swine nucleotide sequences are at least 95% identical tothe human capture reference sequence at orthologous loci (vi)-(x)corresponding to swine loci (i)-(vi), respectively:(vi) exon regions encoding at least one of HLA-A, HLA-B, and HLA-C;(vii) exon regions encoding at least one of HLA-E, HLA-F, and HLA-G;(viii) exon regions encoding at least one of HLA-DR and HLA-DQ;(ix) one or more exons encoding human beta 2 microglobulin (hB2M);(x) exon regions encoding at least one of MIC-A, MIC-B, PD-L1, CTLA-4,EPCR, TBM, and TFPI from the human capture reference sequence,replacing nucleotide sequences in (i)-(v) with said synthetic donorswine nucleotide sequences; andobtaining the swine tissue or organ for xenotransplantation from agenetically reprogrammed swine having said synthetic donor swinenucleotide sequences.

Item 103. The method of item 102, further comprising confirming that thegenetically reprogrammed swine having said synthetic donor swinenucleotide sequences is free of at least the following zoonoticpathogens:

(i) Ascaris species, Cryptosporidium species, Echinococcus, Strongyloidssterocolis, and Toxoplasma gondii in fecal matter;

(ii) Leptospira species, Mycoplasma hyopneumoniae, porcine reproductiveand respiratory syndrome virus (PRRSV), pseudorabies, transmissiblegastroenteritis virus (TGE)/Porcine Respiratory Coronavirus, andToxoplasma gondii by determining antibody titers;

(iii) Porcine Influenza;

(iv) the following bacterial pathogens as determined by bacterialculture: Bordetella bronchisceptica, Coagulase-positive staphylococci,Coagulase-negative staphylococci, Livestock-associated methicillinresistant Staphylococcus aureus (LA MRSA), Microphyton and Trichophytonspp.;(v) Porcine cytomegalovirus; and(vi) Brucella suis.

Item 104. The method of any one of or combination of items 102-103,further comprising maintaining the genetically reprogrammed swineaccording to a bioburden-reducing procedure, said procedure comprisingmaintaining the genetically reprogrammed swine in an isolated closedherd, wherein all other animals in the isolated closed herd areconfirmed to be free of said zoonotic pathogens, wherein the geneticallyreprogrammed swine is isolated from contact with any non-human animalsand animal housing facilities outside of the isolated closed herd.

Item 105. The method of any one of or combination of items 102-104,further comprising harvesting a biological product from said swine,wherein said harvesting comprises euthanizing the swine and asepticallyremoving the biological product from the swine.

Item 106. The method of any one of or combination of items 102-105,further comprising processing said biological product comprisingsterilization after harvesting using a sterilization process that doesnot reduce cell viability to less than 50% cell viability as determinedby a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)-reduction assay.

Item 107. The method of any one of or combination of items 102-106,further comprising storing said biological product in a sterilecontainer under storage conditions that preserve cell viability.

Item 108. A method of screening for off target edits or genomealterations in the genetically reprogrammed swine comprising a nucleargenome of any one of or combination of items 1-49, comprising:

performing whole genome sequencing on a biological sample containing DNAfrom a donor swine before performing genetic reprogramming of the donorswine nuclear genome, thereby obtaining a first whole genome sequence;

after reprogramming of the donor swine nuclear genome, performing wholegenome sequencing to obtain a second whole genome sequence;

aligning the first whole genome sequence and the second whole genomesequence to obtain a sequence alignment;

analyzing the sequence alignment to identify any mismatches to theswine's genome at off-target sites.

Item 109. A synthetic nucleotide sequence having wild-type swine intronregions from a wild-type swine MHC Class Ia, and reprogrammed at exonregions encoding the wild-type swine's SLA-3 with codons of HLA-C from ahuman capture reference sequence that encode amino acids that are notconserved between the SLA-3 and the HLA-C from the human capturereference sequence.

Item 110. The synthetic nucleotide sequence of item 109, wherein thewild-type swine's SLA-1 and SLA-2 each comprise a stop codon.

Item 111. A synthetic nucleotide sequence having wild-type swine intronregions from a wild-type swine MHC Class Ib, and reprogrammed at exonregions encoding the wild-type swine's SLA-6, SLA-7, and SLA-8 withcodons of HLA-E, HLA-F, and HLA-G, respectively, from a human capturereference sequence that encode amino acids that are not conservedbetween the SLA-6, SLA-7, and SLA-8 and the HLA-E, HLA-F, and HLA-G,respectively, from the human capture reference sequence.

Item 112. A synthetic nucleotide sequence having the syntheticnucleotide sequences of both items 109 and 111 or both items 110 and111.

Item 113. A synthetic nucleotide sequence having wild-type swine intronregions from a wild-type swine MHC Class II, and reprogrammed at exonregions encoding the wild-type swine's SLA-DQ with codons of HLA-DQ,respectively, from a human capture reference sequence that encode aminoacids that are not conserved between the SLA-DQ and the HLA-DQ,respectively, from the human capture reference sequence, and wherein thewild-type swine's SLA-DR comprises a stop codon.

Item 114. A synthetic nucleotide sequence having the syntheticnucleotide sequences of: both items 109 and 113; both items 110 and 113;or both items 112 and 113.

Item 115. A synthetic nucleotide sequence having wild-type swine intronregions from a wild-type swine beta-2-microglobulin and reprogrammed atexon regions encoding the wild-type swine's beta-2-microglobulin withcodons of beta-2-microglobulin from a human capture reference sequencethat encode amino acids that are not conserved between the wild-typeswine's beta-2-microglobulin and the beta-2-microglobulin from the humancapture reference sequence, wherein the synthetic nucleotide sequencecomprises at least one stop codon in an exon region such that thesynthetic nucleotide sequence lacks functional expression of thewild-type swine's β2-microglobulin polypeptides.

Item 116. A synthetic nucleotide sequence having wild-type swine intronregions from a wild-type swine MIC-2, and reprogrammed at exon regionsof the wild-type swine's MIC-2 with codons of MIC-A or MIC-B from ahuman capture reference sequence that encode amino acids that are notconserved between the MIC-2 and the MIC-A or the MIC-B from the humancapture reference sequence.

Item 117. A synthetic nucleotide sequence having wild-type swine intronregions from a wild-type swine CTLA-4, and reprogrammed at exon regionsencoding the wild-type swine's CTLA-4 with codons of CTLA-4 from a humancapture reference sequence that encode amino acids that are notconserved between the wild-type swine's CTLA-4 and the CTLA-4 from thehuman capture reference sequence.

Item 118. A synthetic nucleotide sequence having wild-type swine intronregions from a wild-type swine PD-L1 and reprogrammed at exon regionsencoding the wild-type swine's PD-L1 with codons of PD-L1 from a humancapture reference sequence that encode amino acids that are notconserved between the wild-type swine's PD-L1 and the PD-L1 from thehuman capture reference sequence.

Item 119. A synthetic nucleotide sequence having wild-type swine intronregions from a wild-type swine EPCR and reprogrammed at exon regionsencoding the wild-type swine's EPCR with codons of EPCR from a humancapture reference sequence that encode amino acids that are notconserved between the wild-type swine's EPCR and the EPCR from the humancapture reference sequence.

Item 120. A synthetic nucleotide sequence having wild-type swine intronregions from a wild-type swine TBM and reprogrammed at exon regionsencoding the wild-type swine's TBM with codons of TBM from a humancapture reference sequence that encode amino acids that are notconserved between the wild-type swine's TBM and the TBM from the humancapture reference sequence.

Item 121. A synthetic nucleotide sequence having wild-type swine intronregions from a wild-type swine TFPI and reprogrammed at exon regionsencoding the wild-type swine's TFPI with codons of TFPI from a humancapture reference sequence that encode amino acids that are notconserved between the wild-type swine's TFPI and the TFPI from the humancapture reference sequence.

The present invention is described in further detail in the followingexamples which are provided to be illustrative only, and are notintended to limit the scope of the invention.

Example 1 DPF Closed Colony Skin Graft (Monkey Studies)

It has been discovered that skin grafts derived from a DPF ClosedColony, α-1,3-galactosyltransferase [Gal-T] knockout pigs produced inaccordance with the present invention exhibit significantly longerrejection times than skin grafts derived fromα-1,3-galactosyltransferase [Gal-T] knockout pigs but that were notderived from DPF Closed Colony pigs.

Numerous prior studies evaluating rejection time ofα-1,3-galactosyltransferase [Gal-T] knockout pigs (not derived from aDPF Closed Colony) on monkeys show rejection times in the range of 11-13days. See, e.g., Albritton et al., Lack of Cross-Sensitization Betweenalpha-1,3-Galactosyltransferase Knockout Porcine and Allogeneic SkinGrafts Permits Serial Grafting, Transplantation & Volume 97, Number 12,Jun. 27, 2014, (Gal-T-KO skin grafts on recipient baboons fully rejectedby 12 or 13 days); Barone et al., “Genetically modified porcinesplit-thickness skin grafts as an alternative to allograft for provisionof temporary wound coverage: preliminary characterization,” Burns 41(2015) 565-574 (Gal-T-KO skin grafts on recipient baboons fully rejectedby 11 days); and Weiner et al., Prolonged survival of Gal-T-KO swineskin on baboons, Xenotransplantation, 2010, 17(2): 147-152 (Gal-T-KOxenogeneic split-thickness skin grafts on baboons fully rejected by 11days).

The subject invention has been shown in nonclinical studies to performon par and surprisingly better than its allograft comparators, withoutthe inherent disadvantage of inconsistent quality and unreliable andlimited availability. That is, surprisingly, at least Study No. 1 showsskin grafts derived from a DPF Closed Colony,α-1,3-galactosyltransferase [Gal-T] knockout pigs produced in accordancewith the present invention performed better than allograft.

Two recent studies (Study No. 1 and Study No. 2 set out below) byapplicant demonstrate that skin grafts derived from DPF Closed Colony,α-1,3-galactosyltransferase [Gal-T] knockout pigs produced in accordancewith the present invention on monkeys show significantly higherrejection times, in Study 2 longer than 30 days. The geneticallyengineered source animals in this example did not contain any foreign,introduced DNA into the genome; the gene modification employed wasexclusively a knock-out of a single gene that was responsible forencoding for an enzyme that causes ubiquitous expression of acell-surface antigen. The xenotransplantation product in this exampledoes not incorporate transgene technologies, such as CD-46 or CD-55transgenic constructs.

Study No. 1

This study evaluated DPF Closed Colony, α-1,3-galactosyltransferase[Gal-T] knockout porcine xenotransplantation product material comparedto allografts as temporary wound grafts prior to autograft placement incynomolgus monkeys (Macaca fascicularis) in an experimental model offull thickness skin lesions.

Primary end points included screening for porcine endogenous retrovirus(PERV) in the grafts and the recipient as well as evaluation of thexenotransplantation product and allograft rejection and their potentialeffects on ultimate autograft take. Secondary end points includedmicrobiologic and histopathologic analysis of kidney, spleen, liver,lung, grafts, and wound bed tissues collected at necropsy.

Four (4) cynomolgus monkeys were enrolled in this study. Four (4), fullthickness wound beds measuring approximately 2-3 cm×2-3 cm were createdon the dorsal region of each animal on Day 0.

Initially, wounds were treated with either Xenogeneic skin(xenotransplantation product), a split-thickness Gal-T-transgenicporcine xenotransplantation product material, or Allogenic skin(allograft), a split-thickness allograft material, on Day 0.

On Day 15 of the study, the xenotransplantation product and allograftswere removed and replaced with split-thickness autologous skin grafts(autografts), after which the animals were survived to Day 22 of study(with the exception of moribund sacrifice Animals 1001 and 1004).

Microscopic evaluation of full thickness wound beds in a cynomolgusmonkey model treated with xenotransplantation product or allograft andremoved on Day 12 or 15 (FIG. 40A) and survived up to Day 22 (FIG. 40B)demonstrated no evidence of acute tissue rejection with either thexenotransplantation product or allograft comparable to slightly betterperformance overall with the xenotransplantation product test articlewhen compared to the allograft test article, and average to goodautograft performance following pretreatment with eitherxenotransplantation product or allograft test articles. The significantsurvival times of the xenotransplantation product prompted a follow-onstudy (Study No. 2).

Study No. 2

The objective of this study was to evaluate the safety andimmunogenicity of DPF Closed Colony, α-1,3-galactosyltransferase [Gal-T]knockout porcine xenotransplantation product material in cynomolgusmonkeys (Macaca fascicularis).

Primary end points included screening for porcine endogenous retrovirus(PERV) pre- and post-graft placement and evaluation of thexenotransplantation product rejection.

Four (4) cynomolgus monkeys were enrolled in this study. Two (2) 9 cm²full thickness wound beds were created on the dorsal region of eachanimal created on Day 0.

Wounds were treated with split-thickness Gal-T-knockout porcinexenograft material consisting of dermal and epidermal tissue layers.

FIG. 41 shows the longitudinal progression of porcine split-thicknessskin graft used as a temporary wound closure in treatment offull-thickness wound defects in a non-human primate recipient. Left:POD-0, xenograft at Wound Site 2. Right: POD-30, same xenograft at WoundSite 2. FIG. 42 shows POD-30 histological images for: Top, Center: H&E,Low power image of wound site depicts complete epithelial coverage.Dotted line surrounds the residual xenograft tissue. Bottom, Left: H&E,Higher power image of the large inset box. To the right and below thedotted line is the dermal component of the xenograft, with the xenograftdermal matrix indicated by an open arrow. To the left of the dotted lineis the host dermis (black arrow) and the host dermal matrix. Mildinflammation is present and interpreted to be in response to thexenograft test article. Bottom, Right: H&E, higher power image of thesmall inset box. The dotted line roughly demonstrates the junctionbetween the xenograft test article (below dotted line) and new collagentissue (above dotted line), with intact epithelium at the top of theimage. Mild inflammation in response to the xenograft (open arrows) isobserved.

FIG. 43A graphs the total serum IgM ELISA (μg/mL) for all four subjects(2001, 2002, 2101, 2102) during the course of the study. FIG. 43B graphsthe total serum IgG ELISA (μg/mL) for all four subjects (2001, 2002,2101, 2102) during the course of the study. In some aspects, subjectstransplanted with the product of the present disclosure will have serumIgM and IgG levels of less than 20,000 μg/ml each. In some aspects,subjects transplanted with the product of the present disclosure willhave serum IgM and/or IgG levels below or less than 10%, 5%, 3%, or 1%higher than serum IgM and IgG levels measured prior to transplantation.In some aspects, the claimed method may demonstrate an immunoreactivityincidence rate of less than 5%, 3%, or 1% of subjects transplanted withthe product of the present disclosure.

FIG. 44A graphs systemic concentrations of soluble CD40L as measured byLuminex 23-plex at POD-0, POD-7, POD-14, POD-21, and POD-30. FIG. 44Bgraphs systemic concentrations of TGF-alpha as measured by Luminex23-plex at POD-0, POD-7, POD-14, POD-21, and POD-30. FIG. 44C graphssystemic concentrations of IL-12/23 (p40) as measured by Luminex 23-plexat POD-0, POD-7, POD-14, POD-21, and POD-30.

Animals were terminated at 30 or 31 Days, wound sites were collected andfixed in 10% neutral buffered formalin (NBF) or Modified Davidson'sSolution for the testis and epididymis. It should be noted that whilethe animals were terminated at 30 or 31 days due to the study design andfor comparison purposes, the xenotransplantation product of the presentdisclosure is capable of resisting rejection for longer than the studyperiod used in this example.

Microscopic evaluation of full thickness wound beds in a cynomolgusmonkey model treated with xenograft and terminated on Day 30 or 31demonstrated good filling of the wound defect with host and xenografttissue.

Screening for porcine endogenous retroviruses (PERV) and porcinecytomegalovirus (PCMV) was performed separately at specifiedpost-operative intervals via specialized (porcine specific) polymerasechain reaction (PCR) and reverse transcriptase PCR (RT-PCR) testing ofsamples. The porcine xenografts, lysed PBMCS of the recipient, recipientwound bed, and highly perfused organs from the recipients at necropsywere evaluated for presence of porcine cell migration. All tests wereperformed in triplicate with internal controls for DNA and RNA, as wellas assay performance. Microbiologic (bacterial, fungal, viral) assaysand histopathologic analysis of kidney, spleen, liver, lung, xenografts,allografts, wound bed tissues collected at necropsy, and analysis ofperipheral blood were performed to test for xenograft-relatedimmunogenic biomarkers. DNA PCR was performed to test for porcine cellmigration in PBMCs from the cynomolgus monkey model treated with theproduct of the present disclosure for the following samples: (A) (3)full-thickness (xenograft) wound beds, (B) (3) full-thickness(allograft) wound beds; (C) (2) spleen samples; and (D)(2) kidneysamples. There was no evidence of cell migration or zoonotictransmission systemically to the host. The presence of PERV isattributed to the residual pig cells in the wound bed, as verified withporcine MHC controls. Our results suggest that porcine DNA and cells didnot migrate into the circulation of the graft recipients from thegrafts, and likewise PERV or PERV-infected porcine cells did not migratepast the wound bed.

The following Table 4 shows the analysis for porcine cell migration andtransmission:

TABLE 4 Item PSKl7-01 MHC CCR5 No. Sample Analysis PCMV PERV (swine)(Control) PBMC @ End-of-Study Subject # (EoS Date) 1 NHP-1001 (POD-IS) * * * * 2 NHP-1002 (POD-22) Neg (−) Neg (−) Neg (−) Pos (+) 3NHP-1003 (POD-22) Neg (−) Neg (−) Neg (−) Pos (+) 4 NHP-1004 (POD-12)Neg (−) Neg (−) Neg (−) Pos (+) Wound Bed @ End-of- Study Subject #(Test Article) (EoS Date) 5 NHP-1001 (Xenograft) * * * * (POD-IS) 6NHP-1001 (Allograft) * * * * (POD-I S) 7 NHP-1002 (Xenograft) Neg (−)Neg (−) Neg (−) Pos (+) (POD-22) 8 NHP-1002 (Allograft) Neg (−) Neg (−)Neg (−) Pos (+) (POD-22) 9 NHP-1003 (Xenograft) Neg (−) Neg (−) Neg (−)Pos (+) (POD-22) 10 NHP-1003 (Allograft) Neg (−) Neg (−) Neg (−) Pos (+)(POD-22) 11 NHP-1004 (Xenograft) Neg (−) Pos (+)^((A)) Neg (−) Pos ( +)(POD-12) 12 NHP-1004 (Allograft) Neg (−) Neg (−) Neg (−) Pos (+)(POD-12) Spleen @ End-of-Study 13 NHP-1001 Neg (−) Neg (−) Neg (−) Pos(+) 14 NHP-1004 Neg (−) Neg (−) Neg (−) Pos (+) Kidney @ End-of-Study 15NHP-1001 Neg (−) Neg (−) Neg (−) Pos (+) 16 NHP-1004 Neg (−) Neg (−) Neg(−) Pos (+) Key for Table 4: Neg (−) = Negative Pos (+) = Positive *=Test Not Performed or Sample Not Acceptable, due to unrelated, studydesign-related logistical or preservation issue Pos (+)^(A) The woundbed for NHP 1004 (PERV positive) underwent co-culture studies toascertain whether the detected virus present at the interface betweengraft and recipient (host) could infect permissive human cells.Co-culture of the xenograft and recipient wound bed cells withpermissive human cells for PERV infection and replication did notdemonstrate productive infection in the target cells (HEK293), after a23-day culture.

TABLE 5 Banff Grades and Pathologic Component Scores¹ of SkinXenotransplants at POD-30 Surgeon Banff Animal Graft assessment Gradepc² pa³ ei⁴ e⁵ v⁶ c⁷ cav⁸ 2001 1 100% III 3 3 3 1 0 1 0re-epithelialized 2001 2 100% III-IV 3 3 3 2 0 1 0 re-epithelialized2002 1 30% III-IV 3 3 3 3 0 0 0 re-epithelialized 2002 2 30% III-IV 3 33 3 0 0 0 re-epithelialized 2101 1 40% II 3 3 2 3 0 0 0re-epithelialized 2101 2 40% III-IV 3 3 3 2 0 0 0 re-epithelialized 21021 20% III-IV 3 3 3 3 0 0 0 re-epithelialized 2102 2 20% II-III 3 3 3 3 00 0 re-epithelialized ¹Pathologic Component Scores developed by Rosales,et al. ²pc = perivascular cells - number of cells surrounding dermalvessels (venules, capillaries, and arterioles) in deep and superficialdermis; scored on the most involved vessels; pc3 >50 cells/vessel ³pa =perivascular dermal infiltrate area -percent area occupied by the mostinvolved dermal vessels at 40× magnification; pa3 >75% ⁴ei = epidermalinfiltrate - total number of mononuclear cells per four 20x fields; ei3= transepidermal infiltrate, ei2 >20 cells ⁵e = epidermal injury andnecrosis - presence of keratinocyte apoptosis and necrosis; e3 =sloughed, e2 = focal necrosis, e1 - apoptosis ⁶v = endarteritis -mononuclear cells underneath arterial endothelium; scored on the mostinvolved artery; v0 = none ⁷c = capillaritis - maximum number of cellsper capillary cross section; scored on most involved capillaries; c1 -2-4/capillary. c0 = 0-1/capillary ⁸cav = chronic allograftvasculopathy - intimal thickening with luminal reduction; scored aspercent luminal reduction; cav0 = none

The general appearance for all xenotransplants for the course of thestudy was pink, warm to the touch and adherent to the wound bed.Epidermolysis (mild to moderate) was first noted by the surgeon onPOD-14 but the dermis was adherent. Assessment at POD-21 revealed thatthe wound bed was re-granulating and there were signs ofre-epithelialization, such that by POD-30, 20% to 100% of the wound hadbeen re-epithelialized. (Table 5) During the clinical course of theseskin xenotransplants, there was no sloughing of the xenotransplanttissue and exposure of the wound bed.

Hematoxylin and eosin (H&E)—prepared sections containing residual skinxenotransplants and the underlying wound beds, obtained at POD-30, weremicroscopically evaluated by a blinded pathologist. H&E staining showedminimal to moderate inflammatory response. There was ulceration of theepithelia in four out of eight treated sites. The response at the woundsites was characterized by filling of the wound defect with a maturedermal collagen network surrounded by a variable layer of new collagen.This mature collagen network was distinct in appearance from the hostdermis bordering the wound site, and was interpreted to be thexenotransplant dermis. The skin xenotransplants were assessed using asystematic pathologic component scoring and Banff classification⁴⁷. TheBanff classification is useful in categorizing xenotransplant rejection,and it is complemented by the component score approach, providing a morecomprehensive array of clinical thresholds for the diagnosis ofrejection. The results of this assessment and Banff Grades for POD-30are shown in Table 5. The Banff 2007 Working Classification forComposite Tissue Allografts is based on the level of epidermalapoptosis, epidermal infiltrates, and perivascular/dermal infiltrates⁴⁸.The Banff Grades ranged from II (moderate) to IV (necrotizing acuterejection) with most showing Grade III (severe).

TABLE 6 Changes in Serum Cytokines and Chemokines after XenograftTransplantation (pg/mL) Cytokine/ Chemokine POD-0 POD-7 POD-14 POD-21POD-30 sCD40L 1900 ± 7900†± 7700†‡ ± 8600†‡ ± 8500†‡ ± 1000 3100 31004000 5200 IL-1ra 7.6 ± 50† ± 28† ± 66† ± 24† ± 2.8 44 11 83 13 IL-2 29 ±42† ± 37† ± 41† ± 30 ± 11 18 11 9 12 IL-6 0.31 ± 7.3† ± 4.1† ± 8.5† ±3.3† ± 0.6 8.3 2.6 6.3 2.7 IL-8 2500 ± 4200† ± 3700† ± 3900† ± 2500 ±1300 3200 2600 2300 2100 IL-12/23 (p40) 0.6 ± 1.8 ± 26† ± 16† ± 6.7† ±1.0 2.7 22 11 7.7 IL-15 3.1 ± 6.0† ± 7.1† ± 5.0† ± 6.0† ± 1.9 2.0 1.31.3 1.5 MCP-1 360 ± 710† ± 420† ± 460† ± 310 ± 150 540 110 110 120 TGF-α4.5 ± 22† ± 16† ± 5.2 ± 9.9 ± 4.6 11 11 3.6 8.9 POD = Postoperative dayValues are means (n = 4) ± SD †Significant datapoints (p < 0.05)compared to POD 0, student t-test ‡Values include data at the upperlevel of detection (12,000 pg/mL)

As an evaluation of cell-mediated immune response, a total of 23inflammatory and anti-inflammatory cytokines characteristic of initialwound healing processes or those anticipated in an immunologicalresponse to xenogeneic cells were measured. Twelve of the 23cytokines/chemokines assayed were consistently below the level ofdetection throughout the entire study period: TNF-α, IFN-γ, TGF-β,G-CSF, GM-CSF, IL-1-β, IL-4, IL-5, IL-10, IL-13, IL-17, IL-18, andMIP-1-α. VEGF exceeded the level of detection at only three individualtimepoints, and levels of MIP-1-beta were discernable only once (datanot presented). Nine cytokines/chemokines detected over the period ofthe study are listed in Table 6. All cytokines/chemokines shown in thetable were observed to increase above background at POD-7, the first dayof sampling. IL-2, IL-8, MCP-1 and TGF-α peaked at POD-7 and decreasedover time. IL-15 and IL-12/23 (p40) peaked at POD-14, while sCD40L,IL-1ra and IL-6 had an elevated peak at POD-21. In general, all of thefactors showed a return to normal by POD-30 with the exception ofsCD40L, which remained elevated at POD-30. Of interest, levels ofIL-12/23 (p40) were nearly absent until conspicuously elevated onPOD-14, gradually reducing in concentration over the remainder of thestudy.

TABLE 7 Post-Transplant Changes in Binding of Recipient Serum IgM andIgG to PBMC¹ Targets from GalT—KO² Swine Donors Pre/Post IgM⁵ IgG⁶Recipient Transplant³ rMFI⁴ Fold Change rMFI⁴ Fold Change 2001 Pre 8.510.0 16.28 0.0 Post 45.72 4.4 1089.85 65.9 2002 Pre 5.07 0.0 28.29 0.0Post 30.01 4.9 840.64 28.7 2101 Pre 7.92 0.0 16.03 0.0 Post 22.48 1.8730.83 44.6 2102 Pre 6.47 0.0 5.19 0.0 Post 15.49 1.4 372.88 70.8 ¹PBMC= peripheral blood mononuclear cell ²GalT—KO = alpha-1,3galactosyltransferase knockout ³Pre-transplant = POD-0; Post-transplant= POD-30 ⁴rMFI = relative Mean Fluorescent Intensity ⁵IgM =immunoglobulin M ⁶IgG = immunoglobulin G

To assess the production of antibody to xenogeneic after skintransplant, binding of recipient serum IgM and IgG to peripheral bloodmononuclear cell (PBMC) targets from GalT-KO donors was measured by flowcytometry. Serum IgM and IgG antibody levels were analyzed atpre-transplant and at POD-30. In Table 7, the relative mean fluorescentintensity (MFI) and fold increase in binding are summarized for eachrecipient. An increase in anti-xenogeneic IgM and IgG was detected inall animals. Between pre-transplant (POD-0) and post-transplant(POD-30), IgM anti-porcine antibodies increased between 1.4 to 4.9 foldand IgG anti-porcine antibodies increased between 28.7 to 70.8 fold.These results demonstrate a humoral response to non-Gal xenoantigens.

TABLE 8 Data for Postoperative Analysis of Wound Beds (Wound Site 1 and2) PERV Animal Wound copies/500 ng Micro- ID Site (SD) chimerism† QC‡2001 W1 <LOD† − + W2 1495.6 (±521) + + 2002 W1 1518.8 (±21)  + + W2 <LOD− + 2101 W1  527.1 (±134) + + W2 137.8 (±16) + + 2102 W1 <LOD − + W2<LOD − + SD = Standard Deviation, LOD = Limit of Detection, QC = QualityControl †Porcine microchimerism cannot be accurately quantified due tomixture of cells present in wound bed extraction ‡All QC gave a positiveCt , indicating no inhibition

Naïve skin xenotransplants were analyzed for PERV copy number and asexpected, each cell contained copies of PERV A (32±1), B (9±0.1) and C(16±0.1). Sera from the four recipients were evaluated for the presenceof circulating PERV; all samples were found to be negative for PERV poland below the limit of detection. PBMC samples from each of the fourrecipients were also tested for PERV and for microchimerism (i.e., thepresence of circulating pig cells) and were also found negative, at alltime points. Tissues taken at the end of the study (POD-30) wereevaluated for PERV expression and again were found negative. Wound bedsfrom animal 2102 were negative for the presence of PERV and formicrochimerism. (Table 8) For the other animals, either one wound siteor both were positive. This is not surprising due to the direct contactof the wound bed with the xenograft. It is expected that some porcinecells not associated with the graft may have sloughed off or been leftbehind in the process of removal at the end of the study. This isconfirmed by the positive values achieved for the microchimerism assayattributing the PERV signal to porcine cell contamination. Altogether,these results provided no evidence of PERV transmission, consistent withprevious studies.

Example 2

The following example provides a description of a process of harvestingand processing skin from a genetically reprogrammed swine produced inaccordance with the present invention, with the skin to be used as axenogeneic skin product for human transplantation. In some of theseaspects, the xenotransplantation product consists of split thicknessgrafts consisting of dermal and epidermal tissue layers containingvital, non-terminally sterilized porcine cells derived from specialized,genetically reprogrammed, Designated Pathogen Free (DPF), sourceanimals.

In one aspect, the genetically reprogrammed source animal is anygenetically reprogrammed animal described in the present disclosure. Inone non-limiting aspect, the genetically engineered source animals inthis example do not contain any foreign, introduced DNA into the genome;the gene modification includes a knock-out of a single gene that wasresponsible for encoding for an enzyme that causes ubiquitous expressionof a cell-surface antigen. The xenotransplantation product in thisexample does not incorporate transgene technologies, such as CD-46 orCD-55 transgenic constructs.

The process and techniques disclosed herein are but examples, and do notlimit the scope of the invention. It will be fully understood that whilethis example is directed to xenotransplantation skin products, severalof the steps in the following process and aspects of the overallapproach can be applied to other organs or tissues, including, but notlimited to, kidney, lung, liver, pancreas, nerve, heart, intestine, andother organs or tissue. It will be further understood that modificationsto the processes and methods disclosed in this example (includingadditions or omissions of one or more process or method steps) can bemade in relation to the harvesting and processing of other organs ortissue besides skin. This understanding is based in part on the factthat other organs and tissue will have different physicalcharacteristics and so harvesting and processing steps for such otherorgans or tissue will be different from this example in certainpractical ways (e.g., a kidney, heart, liver, lung, or other whole organwill not be cut to size and packaged in a cryovial supported by nylonmesh). Nonetheless, it will be further understood that additions oromissions of one or more process or method steps as applied to each suchorgan or tissue may be made to this example utilizing approaches knownin the art (e.g., a harvested kidney, heart, liver, lung, or other wholeorgan will, in some aspects, be placed in an antipathogen bath orexposed to UV light as described herein for the removal of pathogensfollowing harvest, and placed in one or more closure systems. Forexample, such one or more closure systems could include, but not belimited to, a first closure system (e.g., utilizing an inert materialfor initial closure to surround the organ to prevent the organ fromcoming into contact with or adhering to other materials proximate to theorgan) and/or a second closure system (e.g., a sterile and secure outercontainer that contains the organ and first closure system (if a firstclosure system is utilized)). Such organs within such closure system(s)are configured to be transported to a clinical site as whole organs,stored, protected and transported in temperatures, sterility, and otherconditions to maintain sterility and cell viability for transplantationas described herein at the clinical site.

Animal Preparation

Skin product processing occurs in a single, continuous, andself-contained, segregated manufacturing event that begins with thesacrifice of the source animal through completion of the production ofthe final product.

Xenogeneic skin grafts derived from the genetically reprogrammed sourceanimal is received, with the swine being recently euthanized via captivebolt euthanasia in another section of the DPF Isolation Area. The sourceanimal is contained in a sterile, non-porous bag that is containedwithin a plastic container which is delivered into the DPF IsolationArea and placed in an operating room where the procedure to harvest skinfrom the source animal will occur. All members of the operating teamshould be in full sterile surgical gear dressed in sterile dress tomaintain designated pathogen free conditions prior to receiving thesource animal and in some instanced be double-gloved to minimizecontamination.

The operating area is prepared with materials required for harvestingskin from the source animal prior to decontamination (e.g., 24 hoursprior with chlorine dioxide gas treatment) and prior to the procedure.Dermatome (electronic skin harvesting device, e.g., Amalgatome byExsurco) power supply, and extension cord are sterilized and placed inthe operating area prior to the operation. Any materials not in the roomduring the chlorine dioxide gas treatment (and therefore non-sterile)will be sprayed with 70% ethanol or isopropanol prior to entering theroom.

The source animal is removed from the bag and container in an asepticfashion, for example, a human lifting the source animal from the bag andcontainer using sterilized gloves and/or sterilized device to aidlifting and minimize contamination. The source animal is scrubbed byoperating staff for at least 2 minutes with Chlorhexidine brushes overthe entire area of the animal where the operation will occur,periodically pouring Chlorhexidine over the area to ensure coverage.

The source animal is placed on its right lateral flank and dorsumtowards the operating table leaving the left lateral flank and dorsumexposed. The exposed surface is scrubbed to the extreme visible surgicalborders, and constrained by sterile drapes secured with towel clamps.The source animal is then scrubbed with opened Betadine brushes andsterile water rinse over the entire area of the animal where theoperation will occur for approximately 2 minutes.

This Chlorhexidine and Betadine mixture will sit on the source animalfor approximately 2 minutes, and staff (dressed in sterile dress tomaintain designated pathogen free conditions) will then rinse and drythe source animal with sterile water and sterile gauze. The sourceanimal's hair is removed so as to not impact the membrane or introduceanother element that would degrade the cells. Hair removal is done usingsterilized clippers and/or straight razor in the designated pathogenfree environment immediately post-mortem with a clean blade utilizing achlorhexidine lather. Staff will use the clippers and/or straight razor(lubricated in a sterile bath) to remove any remaining hair on theoperating site, taking care to not puncture the skin. This procedurewill be repeated (scrubbing to shaving) by turning the source animalonto the left lateral flank so as to expose the right side. The sourceanimal will be rinsed with sterile water and dried with sterile towelsand sprayed with 70% ethanol. The source animal will be inspectedvisually by the surgeon to ensure proper coverage of scrubbing. Afterthe sterile scrub and final shaving, the source animal is ready for skinharvest.

Skin Harvesting

Operators will be dressed in sterile dress in accordance with programand other standards to maintain designated pathogen free conditions. Alltissue from the source animal that will be used for xenotransplantationis harvested within 15 hours of the animal being sacrificed.

In one aspect, the source animal is laid on its side on an operatingtable. In this aspect, harvesting is done utilizing a dermatome circularblade, (for example and Amalgatome® SD). As the staff secures the animalin place, the surgeon determines the most appropriate width (e.g., 1, 2,3, or 4 inches) and uses the circular dermatome to remove strips ofsplit thickness skin grafts at a chosen thickness (e.g., 0.50 mm, 0.55mm, 0.62 mm).

By way of further example, the thickness of the skin grafts could rangefrom 0.01 mm to 4 mm, depending on the therapeutic needs at issue. Itwill also be understood that in some aspects a full thickness graft mayalso be utilized harvested with alternative harvesting and graftingprocedures known in the art. Graft sizes can range from 1 cm² to 1000cm² (or approximately 1 ft²). It will be understood that larger graftsizes are also possible depending on the application and harvestingtechnique utilized and size of the source animal. It will be understoodthat for all aspects, other depths could be utilized as well, dependingon the application and needs of the task at hand for therapeutic and/orother purposes.

In another aspect, skin harvesting involves surgically removing a skinflap from the animal first, then the skin flap is placed dermis-sidedown onto a harvest board (e.g., a solid board made of metal, plastic orother appropriate material) set upon on the operating table. In thisaspect, sterile padding material is added beneath the skin flap and ontop of the harvest board, to allow appropriate give for proper dermatomedevice function. The skin flap is then affixed to the harvest boardfirmly with steel clamps. Curved towel clamps are utilized on the sideof the skin flap opposite the clamps until the skin is firm and taut.The surgeon will choose the most appropriate thickness on the dermatomeand adjust per harvest conditions. The surgeon will use the dermatome onthe secured skin flap, with an assistant maintaining tension along thedermatome progress. A second assistant may also provide assistance withskin flap tension, and may use rat tooth forceps to pull the graftproduct emerging from the dermatome.

Grafts are trimmed to desired sizes. By way of example, sizes can be: 5cm×5 cm, with a total surface area of 25 cm² and uniform thickness ofapproximately 0.55 mm; 5 cm×15 cm, with a total surface area of 75 cm²and uniform thickness of approximately 0.55 mm; 8 cm×7.5 cm, with atotal surface area of 60 cm² and uniform thickness of approximately 0.55mm; 8 cm×15 cm with a total surface area of 120 cm² and uniformthickness of approximately 0.55 mm. It will be further understood thatcustomizable sizes (i.e., width, thickness and length) can be createddepending on patient needs, including larger sheets of skin can beharvested for use in xenotransplantation procedures.

The xenotransplantation product is further processed to be free ofaerobic and anaerobic bacteria, fungus, and Mycoplasma. Under sterileconditions in a laminar flow hood in a drug product processing suiteusing applicable aseptic techniques, immediately after, within 1, 2, 3,4, 5, 6, 7, 8, 9, 10 seconds, within 10 seconds to 1 minute, within 1minute to 1 hour, within 1 hour to 15 hours, or within 15 hours to 24hours following harvest, the xenotransplantation product is placed intoan anti-microbial/anti-fungal bath (“antipathogen bath”). With regard toa skin product, this can occur after the skin product is trimmed to theproper dose size and shape (e.g., trimmed to squares, rectangles, orothers shapes of desired size(s))

The antipathogen bath includes ampicillin, ceftazidime, vancomyocin,amphotericin-B placed in a sterile container and the xenotransplantationproducts are diluted as outlined in the following Table 5 and added toRPMI-1640 medium as outlined in the following Table 6. In one aspect,about 10 mL of medium is removed from the bottle before adding the aboveitems.

TABLE 5 Vial Diluent Approx. Vol Approx. Drug Mg Vol Diluent availableconcentration Ceftazidime 1000 10.0 mL Sterile water 10.8 mL 100 mg/mLAmpicillin 2000 10.0 mL Sterile water   11 mL 180 mg/mL Vancomycin 50010.0 mL Sterile water 50 ug/ mL Amphotericin 50  5.0 mL Sterile water 10 mg/mL B

TABLE 6 Volume (mL) Final added to 500 Drug Concentrations mg/500 mLMedia mL RPMI 1640 Ceftazidime 500-2500 mg/L 250-1250 mg 2.5-10 Ampicillin 500-2500 mg/L 250-1250 mg 1-6 Vancomycin 25-125 mg/L 10-75mg  0.25-2   Amphotericin B 40-200 mg/L 20-100 mg  2-10 Total volumeadded 5.75-28  

It will be understood that while this example is directed toxenotransplantation skin products, other organs, including, but notlimited to, kidney, lung, heart, liver, pancreas, and other organs canbe bathed in the antipathogen bath in accordance with the presentinvention. The amounts of combination of drugs and other chemicals, andduration of exposure to such antipathogen bath, are performed tominimize the affect such exposure has on cell viability andmitochondrial activity to achieve both the desired antipathogen resultand minimal manipulation of the xenotransplantation products inaccordance with the present invention.

As an alternative, or in addition to, removing pathogens via theantipathogen bath, the products are made designated pathogen free by aprocess and system utilizing ultraviolet light. In this aspect, theoperator is dressed in sterile dress in accordance with institutionalstandards to maintain designated pathogen free conditions. The operatorwears eye protection safety glasses for ultraviolet light and lasers.

An ultraviolet laser lamp is set up in a laminar flow hood. Each of thefour corners of the lamp is placed on two container lids that arestacked on top of each other, i.e., four pairs of lids are used tosupport the lamp, or other supporting items, able to position the lampin a temporary or fixed position above the working surface of the hood.The distance from the lamp bulbs (2 bulb tubes total) to the floor ofthe hood is approximately 1.5 inches. The entire interior of the hood issprayed with alcohol, e.g., ethanol or isopropanol. The lamp is turnedon and the operator performs a calculation of time for desired exposurebased on lamp specifications, number of bulbs, and distance between thebulbs and the xenotransplantation product.

The operator pours two baths (one chlorhexidine and one alcohol) intotwo separate bowls and places the two bowls under the hood.

A package of new sterilized cryovials is placed under the hood. Cryovialcaps are unscrewed and placed into the chlorhexidine bath. Each cryovial(without cap) is then turned upside down and plunged open ended into thechlorhexidine bath, for one minute each and then set upright to air dry.Thereafter, the exterior of each cryovial is wiped with chlorhexidineand alcohol utilizing sterile gauze. The cryovial caps are removed fromthe chlorhexidine bath and placed on sterile gauze. The open ends ofeach vial were plunged into alcohol bath for 1 minute each and then setaside to air dry.

Xenotransplantation products recently obtained from theharvest/procurement phase in the surgical room are transferred into theproduct processing room, via a one-way entrance into the laminar flowhood. Anything entering the sterile field is wiped down with 70% ethanolprior to transfer to the operator. The operator will have access to allrequired materials in the laminar flow hood: xenotransplantation product(in sterile container), cryovials, 10 mL syringes and needles, phasefreezer holding rack, and pre-cut nylon mesh. Only one size of theproducts is processed at a time to ensure proper control to final vials.The operator is seated at the laminar flow hood in compliance withsterile, aseptic techniques.

When using UV light sterilization, the product is placed under the UVlamp for a desired period of time, e.g., 2 minutes or more, then turnedover to the other side, and put under the UV lamp for the same period oftime, e.g., 2 minutes or more on opposite side. The time period forexposing a given sample to the UV is varied based on the specificbiological agents or the types of biological agents to be sterilized,e.g., as shown in the following Table 7:

TABLE 7 Type of UV-C Dosage Sterilization Biological (uW sec/cm²) timeBiological Agent Agent for 90% sterilization (sec)* Penicillium spp.Fungus 224,000 1800 Aspergillus flavus Fungus 34,900 300 Aspergillusniger Fungus 31,500 250 Yeast Fungus 4000 30 Influenza A Virus 1900 15HIV-1 Virus 28,000 220 Vaccinia Virus 1500 10 Escherichia coli Bacteria2000 20 Staphylococcus Bacteria 6600 50 aureus Bacillus subtilisBacteria 6800 50 Mycoplasma spp. Bacteria 8400 70 Pseudomonas Bacteria2200 20 aeruginosa *Using a UV-C intensity of 125 uW/cm²

With regard to other whole organs, product yield will typically dependon how many of each such whole organ a given source animal may have(e.g., one liver, two lungs, two kidneys, one heart, one pancreas and soforth).

It will also be understood that while this example is directed toxenotransplantation skin products, other organs, including, but notlimited to, kidney, heart, lung, liver, pancreas, and other organs canbe exposed to ultraviolet light and made designated pathogen free inaccordance with the present invention. The UV exposure dosages,intensity, and duration of exposure to such ultraviolet light, areperformed to minimize the affect such exposure has on cell viability andmitochondrial activity to achieve both the desired antipathogen resultand minimal manipulation of the xenotransplantation products inaccordance with the present invention.

Manufacturing Process Generally

Through the continuous manufacturing event, source animals are processedinto aseptic xenotransplantation products. Several items are involved inthe manufacture of the product relating to the source animals,including, but not limited to:

-   -   a) care and husbandry of the source animals (including, as        described herein, providing certain vaccinations, carefully        maintaining and analyzing pedigree records, performing proper        animal husbandry, and maintaining the animals in isolation        barrier conditions);    -   b) product manufacturing (including, as described herein,        processing the source animals into the subject product from        euthanizing to harvest);    -   c) analytical testing of the source animals (including, as        described herein, screening for adventitious agents including        parasitology, bacteriology, and virology assays);    -   d) analytical testing of the source animals (including, as        described herein, confirming the source animal is an        alpha-1,3-galactotransferase knockout or has other        characteristics that are desired for a given application); and    -   e) analytical testing of the source animals (including, as        described herein, viral assay for Endogenous Viruses (PERV)).

Several items are also involved in the manufacture and release testingof the resulting products, including, but not limited to:

-   -   a) product manufacturing (including, as described herein,        processing the drug product, storing the drug product, and        releasing the drug product);    -   b) analytical testing of the drug product (including, as        described herein, viability testing (via, e.g., MTT assay)),    -   c) sterility testing (including, as described herein, aerobic        bacteria culture, anaerobic bacteria culture, fungal culture,        Mycoplasma assay, endotoxin test, USP <71>)),    -   d) adventitious agent testing (including, as described herein,        PCR Assay for e.g., Endogenous Viruses (PERV)); and    -   e) analytical testing of the drug product (including, as        described herein, histology).

For skin, the quantity of product yield from each animal can varydepending on the size of each animal. By way of example, some animalscould yield between 3,000 and 6,000 cm² in product. In one aspect, asingle batch of skin product is harvested from a single source animal ina continuous process. A batch description of the xenotransplantationproduct is provided in Table 8 and batch formula for thexenotransplantation product is provided in Table 9.

TABLE 8 Batch Size Product (strength) Lot Size Xenotransplantationproduct Drug Product, 200 Units (180-220) Dosage Strength 1 (7.5 grams,25 cm²) (1.5 kgs per lot) (1.35 kg to 1.65 kg) Xenotransplantationproduct Drug Product, 67 units (60-75) Dosage Strength 2 (22.5 grams, 75cm²) (1.5 kgs per lot) (1.35 kg to 1.65 kg)

TABLE 9 Batch Formula Nominal Nominal Component Amount per Vial Amountper Lot Xenotransplantation  25 cm² 200 Units product Drug SubstanceDosage Strength 1 CryoStor 7 ml 1.4 L Nylon Mesh  60 cm² 1200 cm² TotalBatch Size  7.5 grams 1.5 kgs Xenotransplantation  75 cm²  67 Unitsproduct Drug Substance Dosage Strength 1 CryoStor 5 ml 350 ml Nylon Mesh180 cm² 3600 cm² Total Batch Size 22.5 grams 1.5 kgs

Prior lot testing is performed under good laboratory practice (“GLP”)conditions to ensure process sterility is maintained consistently.Assurance of sterility of the final product is determined prior tomaterial release and clinical use. Prior to validation for humanclinical use, all xenotransplantation products will meet certainacceptance criteria, including as described herein. The final drugproduct control strategy and analytical testing is conducted at theconclusion of the manufacturing process prior to release for clinicaluse. Results of the required analytical tests will be documented via adrug product certificate of analysis (COA) that is maintained with amaster batch record pertaining to each lot of xenotransplantationproducts.

Source animal sample archives are generated and maintained throughprocurement of tissue samples of lung, liver, spleen, spinal cord,brain, kidney, and skin. These tissues are collected for source animaltissues for testing, archive, and stored for potential future testing.Archived samples of source animal tissue and bodily fluids should bestored at minus (−) 70 degrees Celsius or lower, as appropriate forpreserving the sample. In other aspects, fixed samples can be maintainedat room temperature. Appropriate tissue samples should be collected forformalin fixation and paraffin-embedding and for cryopreservation fromsource animals at the time the live cells, tissues, or organs areprocured. Cryopreservation should be at least ten 0.5 cc aliquots ofcitrated- or EDTA-anticoagulated plasma; five aliquots of viableleukocytes (1×107/aliquot, for subsequent isolation of nucleic acids andproteins or for use as a source of viable cells for co-culture or othertissue culture assays.

Product Processing Following Harvesting

The previously harvested and minimally manipulated xenotransplantationskin product (here the skin integrity being minimally manipulated dermaland epidermal tissue layers with standard cellular morphology andorganization) enters the separate, adjacent room with positive pressureabove that of the surgical suite, designated as the Class 10,000 (ISO-7)product processing room.

The operating room will be setup per operating preparation proceduresand the operating personnel will be dressed in Tyvex suits for fume hoodwork. If requested, an assistant will also be dressed in a Tyvex suit.Gowning and Dressing is done with aseptic techniques. Gloves and sleeveswill be sprayed with alcohol if needed. The ABSL-2 laminar flow hood,having been prior sterilized via gaseous chlorine dioxide sterilizationprocess, will be sprayed with alcohol, e.g. 70% ethanol, and the laminarflow exhaust will be initiated. Utilizing aseptic techniques, previouslysterilized via autoclave, surgical instrument, cryovials, cryotray,flasks, syringes, needles, additional containers, and all processingequipment will be placed within the laminar flow hood. Exteriorpackaging is sprayed with alcohol prior to being transferred to theoperator.

As described herein, prior to operation, nylon mesh graft backing shouldbe cut into squares of appropriate size for the dosage levels, sealed inan autoclavable pouch, and sterilized via steam. Exterior of pouch willthen be sterilized with 70% ethanol and placed in the fume hood.Exterior package of 10 mL Cryovials will be decontaminated with 70%ethanol and placed into the fume hood. Sterile, autoclaved surgicalinstrument package should be sprayed with 70% ethanol and transferred tothe operator.

Sterile syringes and needles should be sprayed with 70% ethanol andtransferred to the operator. Graft tissue recently harvest form theporcine donor will be transferred to the hood. Anything entering thesterile field is wiped down with 70% ethanol prior to transfer to theoperator. Operator will have access to all required materials in thefume hood: Grafts (in sterile container), Cryovials, 10 mL syringes andneedles, Phase Freezer holding rack, and cut Nylon mesh. Operator shouldbe seated at the fume hood with in compliance with sterile, aseptictechnique.

Referring to FIG. 45, each cryovial will be sterilized and labeled inadvance to reduce processing time and unnecessary material exposure toDMSO prior to cryopreservation. Pans containing each xenotransplantationproduct and the RPMI 1640 Tissue Culture Media at room temperature withantibiotics (e.g., antipathogen bath) is placed under the laminar flowhood. The products had been bathing in the anti-pathogen bath for notless than 30 minutes to sterilize the xenotransplantation product.

In one aspect, when using UV light sterilization, the cryovials aresterilized using the UV lamp as described above. After the product isinserted into each vial, each new cap is placed on each new vial andscrewed on securely. Each vial is placed under the lamp and periodicallyrolled for desired even exposure to light on the exterior of the vial.The vials are placed inside a glass jar that has an interior that hasbeen previously sterilized and the exterior is sterilized by theoperator with alcohol and chlorhexidine, including threads and caps.Vials are wiped down with alcohol and are placed into glass jars. Theexteriors of the glass jars are drenched with alcohol outside of thehood. Under the hood, the operator bathes the glass jar lids and plungesthe open ends of the jars into alcohol and wipes the exterior of thejars with alcohol (and optionally chlorhexidine) including threads ofthe jar. The vials are wiped with alcohol utilizing gauze and placedinside each glass jar with an instrument. The lids of the glass jars arethen secured and the jars are handed to the assistant. Frequently and ona periodic basis throughout these processes, the assistant sprays theoperator's gloves and arms with alcohol.

In this example, the xenotransplantation skin product, which was cut toform in the surgical suite with sterile scissors and was trimmed with10-blade scalpel, will be re-measured with a sterile, stainless steelruler to verify technical specifications and dimensions have been met.The xenotransplantation skin product is visually inspected to ensure norips, tears, observable defects, or excessive or insufficient thicknessare present.

Under the laminar flow hood the operator will use forceps to take asingle xenotransplantation skin product from the antipathogen bath andplace it upon a piece of nylon mesh that has been previously cut to fitthe cryovial, centered on the nylon mesh, with the dermis side incontact with the mesh (e.g., dermis side down), taking 1 minute for eachproduct (understanding the time could be less or more, and up to 5minutes for each product). It will be understood that the sterile nylonmesh packaging component is utilized, among other things, to support thexenotransplantation product and prevent self-adhesion of thexenotransplantation product when rolled.

It will be further understood that the sterile nylon mesh packagingcomponent can be of any dimension that would allow thexenotransplantation product to be placed onto the sterile nylon meshpackaging component and fit within the two dimensional surface area(i.e., the length and width not including the thickness) of the sterilenylon mesh packaging component (e.g., the two dimensional area dimensionof the xenotransplantation product would be less than the twodimensional area dimension of the sterile nylon mesh packagingcomponent).

It will be further understood that the dimensions of the sterile nylonmesh packaging component would be sized in accordance with thexenotransplantation product size and dosage. For example, the sterilenylon mesh packaging component is 8 cm×7.5 cm (60 cm²) to fit a 5 cm×5cm xenotransplantation skin product (25 cm²) (7.5 grams) utilizing 7 mlof cryoprotective media when placed in the cryovial. It will be evenfurther understood that the dimensions of the sterile nylon meshpackaging component is 8 cm×22.5 cm (180 cm²) to fit a 5 cm×15 cmxenotransplantation skin product (75 cm²) (22.5 grams) utilizing 5 ml ofcryoprotective media when placed in the cryovial.

Unintentional adhesion of epidermal or dermal regions of thexenotransplantation skin product during packaging may disrupt theintegrity of the xenotransplantation skin product and potentially reduceits therapeutic viability. Inclusion of the sterile nylon-mesh packagingcomponent is intended to provide internal physical support to andprevent self-adhesion. The sterile nylon-mesh packaging component is notbiologically or chemically active and does not directly impact themetabolic activity or efficacy of the xenotransplantation skin productitself.

During the course of numerous experiments, including the monkey studiesdescribed in Example 1 herein, use of this sterile nylon-mesh packagingcomponent has never been observed to cause an adverse, undesiredreaction with the xenotransplantation product, or degrade andcontaminate the final xenotransplantation product causing adversereactions or outcomes to the recipient. The sterile, nylon-meshpackaging component is not used in the grafting procedure. Followingcryopreservation and thawing, and prior to use of thexenotransplantation product, it is discarded. Thus, selection of thespecific material and associated specifications were carefully chosenfor the given application. Medifab 100-Micron Nylon Mesh (Part#03-100/32-Medifab) is manufactured per cGMP standards, and was selectedbecause of its physical characteristics and certified acceptability forhuman, clinical use.

Under the laminar flow hood, the operator will then tightly roll thiscombination of xenotransplantation product and nylon mesh packagingcomponent and place the combination within a cryovial (e.g., 10 ml vial)taking 1 minute for each product (understanding the time could be lessor more, and up to 5 minutes for each product). In this aspect, the meshmaterial is rolled to ensure that the vertical height of the cylinder is8 cm and uniformly fits within the 10 ml cryovial (e.g., 10 cm lengthand 17 mm diameter) and once completed, can be secured with a threadedseal cap. The mesh material is oriented such that the protective meshmaterial is on the exterior of the xenotransplantation product, and thatonce the rolled is complete there is no exposed or visiblexenotransplantation material and it is fully encased in the protectiveinsert. The intrinsic tensile and material properties of the sterilenylon-mesh packaging component are homogenous, and the inelasticity orstiffness of the material causes it to expand to fill the volume of thecryovial. Thus, regardless of the initial “roll-density”, the materialwill uniformly loosen and is therefore standardized.

Under the laminar flow hood the operator will then use a sterile syringeto draw up enough sterile cryoprotective media (e.g., 5-7 ml of themedia with 5% dimethyl sulfoxide (DMSO) (Cryostor CS5, BioLifeSolutions)) to fill the cryovial until the skin product roll is fullyimmersed, ensuring that the combination of xenotransplantation skinmaterial, mesh backing, and cryoprotectant media is flush with the 10 mlfill line, taking 1 minute for each product (understanding the timecould be less or more, and up to 5 minutes for each product).

Under the laminar flow hood, the operator will seal the cryovial withthe threaded cap. The identity of the contents and label information areconfirmed by the operator. Labels are prepopulated and applied to theexterior of the cryovials containing the product in advance of theproduct processing.

It will be understood that the preparation of the xenotransplantationproducts and packaging components described herein could be in the formof therapeutic dosages. For example, the xenotransplantation drugproduct consists of:

-   -   q. Xenotransplantation split-thickness skin Drug Substance    -   r. Primary Container Closure System which includes        -   i. Primary Packaging Component: a sterile, clear,            polypropylene 10 ml cryovial with threaded seal-cap        -   ii. Sterile nylon-mesh packaging component        -   iii. Cryoprotective media packaging component            The indicated dosage of Xenotransplantation product is 300            mg of vital, metabolically active, porcine            xenotransplantation drug substance per cm², with a constant            thickness of 0.55 mm. Example formulations include:    -   s. Dosage Strength 1: a 25 cm² split thickness skin graft, with        uniform thickness of 0.55 mm, which weighs approximately 7.5        grams.    -   t. Dosage Strength 2: a 75 cm² split thickness skin graft, with        uniform thickness of 0.55 mm, which weights approximately 22.5        grams.

An example xenotransplantation drug product primary packaging componentis a sterile, clear, polypropylene 10 ml cryovial with threadedseal-cap. For example, the Simport Cryovial, T310 (10-ml) ismanufactured by Simport Scientific. This product is composed of medicalgrade resin that is BPA free, Heavy Metal Free, and LATEX Free and meetsUSP Class VI limits.

A nylon-mesh packaging component is utilized during thexenotransplantation drug product manufacturing process. The preparedxenotransplantation drug product is placed on sterile nylon-meshpackaging component (e.g., Medifab 100-Micron Nylon Mesh) that has beenpreviously trimmed to the following dimensions:

-   -   u. Dosage Strength 1: 7.5 cm in width by 8 cm in height; total        area of 60 cm²    -   v. Dosage Strength 2: 22.5 cm in width by 8 cm in height; total        area of 180 cm²

A cryoprotective media packaging component is also utilized during thedrug manufacturing process. The xenotransplantation drug product isimmersed in the following volumes of cryoprotective media packagingcomponent prior to cryopreservation:

-   -   w. Dosage Strength 1: 7 ml of Cryostor CS5 (containing 5% DMSO).    -   x. Dosage Strength 2: 5 ml of Cryostor CS5 (containing 5% DMSO).

With regard to the assurance of saturation of cryoprotective media, theindicated amount of CryoStor CS5 media (per Dosage Strength) is appliedvia 10 ml syringe with the cryovial (such as a type of cryovial shown inFIG. 46) in the vertical position, under a laminar flow hood (ISO-5, FEDSTD 209E Class 100 conditions) Cryomedia fills the voided space(s), andgravity ensures that the fill-process begins from the base of thevertically oriented cryovial towards the fill line at the apex. Volumeis added until it reaches the manufacturers demarcated 10 ml fill line.Filling the vial in this manner also facilitates the removal of airbubbles. Once complete, the threaded cap is sealed. Visual and physicalverification of saturation and fill is accomplished, ensuring thatcontents the xenotransplantation product are unable to shift internally.

Cryopreservation

Product materials will be placed in the appropriate freezer rackcontaining cryovials with product as described above, and placed in acertified, Q-A control rate-phase freezer. Using a certified, Q-Acontrol rate-phase freezer, the entire product is cryopreserved via onestandardized control-rate freezing process:

-   -   y. Starting at 4° C., internal chamber and sample temperature        probe will lower at a rate of 1° Celsius per minute until a        temperature of −40° C. is achieved.    -   z. Once temperature of −40° C. has been reached in a controlled        rate, control-rate freezer sample temperature probe should lower        rapidly from −40° C. to −80° C.    -   aa. Material is then transferred to a GLP certified, −80° C.        freezer until use.        Taking 40 minutes per batch time from room temperature to        −80° C. (understanding the time could be less or more, and up to        2 hours). In some aspects, penetrative cryoprotectants such as        DMSO, may be used to protect morphology and tissue structure,        and retain metabolic activity levels comparable to that of fresh        skin. In some aspects, cryopreservation may alternatively or        additionally include one or more of glycerol, gentamicin,        Nystatin, L-glutamine, and other processing solutions. In some        aspects, β-lactam antibiotics are not used.

Inclusion of the cryoprotective-media packaging component is intended tosupport cell survival during the freeze-thaw cycle required for thexenotransplantation product. Failure to include the cryoprotective mediapackaging component of xenotransplantation product during packaging maydisrupt the integrity of the xenotransplantation product or impede thecryopreservation process, and may potentially reduce thexenotransplantation product viability below acceptance criteria.Cryopreservation of the xenotransplantation product without inclusion acryoprotective media results in destruction of biologically active cellscontained in the xenotransplantation product. Rapid formation of icecrystals and disruption of cellular membranes and mitochondrialorganelle barriers occurs during the freezing process, and thedimethyl-sulfoxide ingredient acts to displace intracellular fluid.Thus, the cryoprotective media reduces the formation of such icecrystals and rapid, disruptive increase in total cellular volume thatwould negatively impact the cellular viability and, thus, the efficacyof the Drug Product.

During the course of a number of experiments, including the monkeystudies in Example 1 herein, use of this cryoprotective-media packagingcomponent has never been observed to cause an adverse, undesiredreaction with the xenotransplantation product, or degrade andcontaminate the final xenotransplantation product causing adversereactions or outcomes to the recipient. Thus, selection of the specificmaterial and associated specifications were chosen to meet appropriatestandards necessary of a xenotransplantation product intended for human,clinical use. This including identifying a cryoprotective media withminimal, subclinical levels of DMSO, one that would satisfactorilyperform without the need for inclusion of an additionalxenotransplantation material (porcine serum) in the formulation. Thecryoprotective media-packaging component is not used in the graftingprocedure. Upon thawing, and prior to use of the xenotransplantation fortherapeutic uses including as a drug product, it is discarded. CryoStorCS5 is manufactured per cGMP standards and was selected because of itscertified acceptability for human, clinical use.

Shipping to Clinical Site

Shipping the product to the clinical site should be done to maintain thexenotransplantation skin product material at −80° C. storage condition.One example shipping container is the EXP-6 Standard Dry Vapor Shipperhaving an extensive, having the following specifications:

-   -   Dynamic Holding Time 10 Days    -   Holding Temperature −150° C. or Colder    -   Core Technology Dry Vapor Liquid Nitrogen    -   Specimen Chamber 2.8″ (71 mm) Diameter    -   11.5″ (292 mm) Depth    -   Weight Dry 9.7 lbs/4.4 kg    -   Charged 18.3 lbs/8.3 kg    -   Domestic Dimensional 21.07 lbs/9.56 kg    -   International Dimensional 24.87 lbs/11.28 kg    -   Outer Box 12″×12″×22″    -   (305×305×559 mm)        Aspects of the shipping process are also shown in FIG. 47        including, but not limited to, (1) cryopreservation storage; (2)        xenotransplantation product in cryovial and media as described        herein while in cryopreservation storage; (3) cryovial placed in        dry vapor shipping container (or secondary closure system); (4)        container and vial shipped via courier; (5) xenotransplantation        product controlled and monitored at delivery location (can last        at least 10 days at minus (−) 150 degrees Celsius or        colder); (6) xenotransplantation product in cryovial and media        as described herein removed from container/secondary closure        system; (7) xenotransplantation product in cryovial and media as        described herein placed in freezer at location being stored at        −80° C.

Clinical Site Preparation

In one aspect, the drug product arrives at the clinical site as acryopreserved xenotransplantation product. Prior to use, thexenotransplantation product must be thawed in a 37° C. water bath,removed from the vial and washed in a series of 3 sterile 0.9% salinebaths at room temperature.

For the thawing process, sterile equipment and aseptic techniques areused:

-   -   a) Prepare 200 mL of normal saline into each of three 500 mL        sterile, surgical bowls.    -   b) Place the unopened cryovial with the skin product in water        bath having a temperature of about 25° C. In some embodiments,        the temperature is about 37° C.    -   c) In the bath, swirl gently for approximately 5 minutes or        until tissue is mobile within the cryovial, taking care to        minimize unnecessary exposure time the xenotransplantation skin        product tissue is suspended in the thawed DMSO as much as        possible.    -   d) Open the cryovial and use sterile forceps to quickly remove        tissue and mesh to transfer into a bowl of normal saline.    -   e) Using sterile forceps, ensure tissue is fully submerged in        saline for 15 seconds, agitating by swirling gently to maximize        coverage. The underlying, supportive mesh material should be        separated from the skin xenotransplantation skin product        material. Use a second pair of sterile forceps to separate if        necessary. Mesh can be left in the bowl, or discarded.    -   f) Using sterile forceps, transfer the skin into a second bowl        wash. Submerge fully and gently swirl for 15 seconds; this is a        serial dilution or “rinse”.    -   g) Repeat the previous step, using sterile forceps to transfer        the skin into a third wash of normal saline. Submerge fully and        gently swirl for about 15 seconds.    -   h) The entire duration of the rinse process should be completed        within 60 seconds to minimize unnecessary exposure time the        product is suspended in thawed DMSO in order to maximize product        efficacy.    -   i) Tissue is now thawed, rinsed, and ready for application.        Leave in normal saline until use, not to exceed 2 hours at about        25° C.

After the complete, thaw and rinse process is complete, thexenotransplantation product is ready for placement on the wound site.Serial washes in saline, once thawed provide ample dilutive solvent toremove the residual cryoprotectant (5% DMSO solution, CryoStor CS5) andreplace the intracellular fluid levels to normal homeostatic conditions.Such dilution and use of a cryoprotective media containing asub-clinical level of DMSO ensures that any minimal, residual DMSOremaining on the xenotransplantation skin product material post-thawwould be non-appreciable and would be highly unlikely to be clinicallysignificant. This process also ensures retention of the maximum amountof metabolically active cells, and thereby maximizing the efficacy ofthe xenotransplantation product.

Example of Thawing. Following is one example of a thawing procedure fora xenotransplantation product. Thawing can occur in a BioSafety Cabinetwith operator in sterile gloves as follows: (i) prepare 200 mL of Normalsaline into each of three 500 mL surgical bowls; (ii) prepare the waterbath by wiping it clean with chlorhexidine then spraying it down with70% ethanol; (iii) after the ethanol has dried add sterile watersolution into the water bath and heat to 37° C.+/−2° C.; (iv) thexenotransplantation drug product is in a double bag, leave it unopenedand place it into the 37° C. water bath; (v) swirl gently forapproximately 5 minutes or until the tissue is mobile within thecryovial; (vi) minimize the time the tissue spends in thawed DMSO asmuch as possible; (vii) spray the outside bags with ethanol and removethe vial from the outer bags and spray the xenotransplantation drugproduct cryovials with 70% ethanol before placing into BiosafetyCabinet; (viii) unscrew the cryovial and use forceps to quickly removetissue and mesh to transfer into a bowl of normal saline; (ix) useforceps to ensure tissue is fully submerged in saline for 60 seconds,agitating by swirling gently to maximize coverage; (x) the mesh shouldbe separated from the skin, using a second pair of forceps to separateif necessary; (xi) the mesh can be left in the bowl, or discarded; (xii)using forceps transfer the skin into the second bowl wash; (xiii)submerge fully and gently swirl for 60 seconds; (xiv) using forcepstransfer the skin into the third bowl wash and submerge fully and gentlyswirl for 60 seconds. Tissue is now thawed and ready for application.Keep it moist with sterile saline in a sterile pan.

The process of rolling the inert, nylon mesh backing and thexenotransplantation skin product results in uniform “roll-density” ofthe xenotransplantation product. All mesh materials are cut to uniformdimensions, according to the prescribed dimensions for the givenapplication, and are obtained from the same material lot, thus affordinguniform material properties for all units of the skin productmanufactured within a specific lot.

The intrinsic tensile and material properties of the nylon mesh insertare homogenous, and the inelasticity or stiffness of the material causesit to expand to fill the volume of the primary container closure system(cryovial). Thus, regardless of the initial “roll-density”, the materialwill uniformly loosen and is therefore standardized.

The indicated amount of CryoStor CS5 media (per Dosage Strength) isapplied via 10 ml-syringe with the cryovial in the vertical position,under Class 100, ISO5 conditions within an ABSL-2 laminar flow hood.

Cryomedia fills the voided space(s), and gravity ensures that thefill-process begins from the base of the vertically oriented cryovialtowards the fill line at the apex. Volume is added until it reaches themanufacturers demarcated 10 ml fill line. Filling the vial in thismanner also facilitates the removal of air bubbles.

Once complete, the threaded cap is sealed. Visual and physical assuranceof saturation and fill is accomplished by the shaking the skin productensuring that contents are unable to shift internally. Aspects of thecryovial are also shown in FIG. 46, with aspects that can include, amongother things, 10 ml volume, size of 17 mm×84 mm, vertical ribsfacilitating cap removal, silicone washer, cap and tube made of the samepolypropylene material with the same coefficient of expansion ensuringseal at all temperatures, 1 and ¼ turn thread design, thick wall, largewhite marking area, and round bottom allowing for ease of emptyingcontents.

Aspects of the secondary closure system is shown in FIG. 48, withaspects that can include, among other things, Tyvek-1073B medical gradeconstruction, 5 inches wide×12″ high, storage ability of 15 cames or 2cryovial boxes, holding temperature of −150 degrees Celsius or colder,utilization of dry vapor liquid nitrogen, IATA rated 10 days of dynamicholding time under normal shipping conditions, specimen chamber diameterof 2.8 inches (71 mm), specimen chamber depth of 11.5 inches (292 mm),dry weight of 9.7 lbs/4.4 kg, charged weight of 18.3 lbs./8.3 kg,domestic dimensional weight of 21.07 lbs./9.56 kg, internationaldimensional weight of 24.87 lbs./11.28 kg, outer box dimensions of12″×12″×22.″

No additional or external impurities in the product are anticipated tobe present since processing involves only the minimal mechanicalmanipulation of the product, and no other chemical or biological agentsare introduced during this closed process. Acceptance criteria testingrequired for use of the source animals for the product manufacturingprocess is conducted as described herein and documented via the DrugProduct COA. The final product is evaluated for viral adventitiousagents as described herein.

In terms of shelf life, continuous storage of the xenotransplantationproduct as described support a shelf life long-term stability(cell-viability) of up to at least 7 years (in one embodiment is a shelflife of 6 months) when stored continuously at −80° C. The shelf-lifeduration of continued cryopreservation of the xenotransplantationproduct with of at least 7 years. Table 10 shows stability time pointsthat the xenotransplantation product will be tested.

TABLE 10 Stability Study Time Points Time points (Months) Assay 0 12 2436 60 Histology A B B B B Sterility A B B B B Endotoxin A B B B BViability A B B B B A = initial product release testing B = stabilitytesting for Xenotransplantation product

In accordance with one aspect, following in Table 11 are items that canbe utilized in a certificate of analysis and release.

TABLE 11 Test Results Test Method Acceptance Criteria Results AppearanceVisual Clear, colorless to slightly Conforms Inspection yellow liquidwith no visible particulates pH TM5110 7.5 to 7.7 7.6 USP <791>Metabolic TMSlOO Cell viability is 75% to 87 Activity 200% of cellspreserved Assay in the internal standard at Day 1 recovery followingpreservation. Endotoxin Kinetic s 0.5 EU/ml Conforms Chromogenic USP<85> Sterility Membrane Sterile Conforms Filtration USP <71>Identification TMSlll FT-IR Conforms to CryoStor Conforms CSS ReferenceStandard Osmolality TM 5112 1360-1390 mOsm/kgH20 1388 USP <785> SpecificTM5114 1.055-1.063 1.059 Gravity DMSO Gas 4.0%-7.0% 5.0 ContentChromatography (FID)

Example 3

Porcine skin shares fundamental properties with human skin andrepresents a potential alternative to human cadaver skin grafts fortemporary coverage of severe burns. The impact of extendedcryopreservation on porcine grafts on graft viability, graft take, andbarrier function was examined in a study using a model of MHC matchedand mismatched MHC Class II skin transplants.

Cellular viability was assessed using formazan-MTT and the biologicalproperties of the grafts, were assessed by grafting on swine recipients.To complement the in vivo clinical assessments, histologic, andmorphologic analyses, a series of MTT-reduction assays were performed toevaluate the residual viability of porcine grafts after cryopreservationand long-term storage. Mitochondria reduce MTT into a formazanmetabolite, which can be observed as purple hue. Harnessing thisphenomenon, an analysis of changes in optical density values measured bya spectrophotometer, or an interpolation of the quantities of formazanproduced against standard curves, can provide differential assessmentsof cellular viability, between experimental samples and positive andnegative controls. There were 2 cohorts of 2 animals each (total, N=4)based upon the MHC match and each swine received 4 grafts: one autograftand three allografts of identical MHC-profiles. Grafts were clinicallyassessed for graft-take, adherence, and time to graft rejection.Rejection was also assessed histologically via the Banff grading scale.

Direct comparisons between otherwise equivalent materials yieldmeaningful, differential times of survival, based solely on duration ofstorage, holding all other factors constant. Side-by-side, in vivoevaluations are performed between equivalent grafts, preserved inidentical fashion, stored for periods of 15 minutes versus 7 years.Clinical gross assessments and photographs, paired with independenthistological assessments, determine whether any appreciable differencesin graft survival exist relative to the length of time in the frozenstate. In tandem, separate in vitro assessments of graft viability,quantified by MTT-reduction assays, characterize the metabolic activityof cells post-cryopreservation and various storage terms. Further,independent histomorphological analysis, using standard histological(H&E) staining, provides evidence as to whether these processes causeobservable changes to the graft material at a structural level. Thisstudy advantageously used materials that had been stored, uninterrupted,for such a time, along with the associated surgical records andstandardized institutional protocols. Further, processing methods andprotocols between the comparative groups were standardized, andidentically applied, with respect to cryopreservation and thawingprotocols, reagents, and methods employed. Combined, this allowed forisolated, side-by-side evaluation of duration of storage, and alleviatedthe need to model or extrapolate findings, or otherwise use normativepredictive methods. Furthermore, the use of MHC-matched and Class IImismatched donor-recipient pairs in this model of allogeneic skintransplantation served as internal controls to both confirm the identityof the tissues obtained seven years earlier, and the veracity of thesurgical notes and documentation. Further, equivalent behavior exhibitedby the allografts also demonstrates that the antigenicity of the graftswas not altered as a result of the duration of storage.

There were no technical failures; all grafts adhered to their respectivewound beds and re-vascularized. In cohort 1 (MHC-matched donor-recipientpair), all grafts remained adherent, and appeared uniformly healthy atpostoperative day (POD) 12 (FIG. 49A), but at POD-14, signs of necrosis,progressive erythema and loss of adherence were observed (FIG. 49B).Clinical assessment of the 6 grafts in cohort 1 showed rejection atPOD-14 to 18. In cohort 2, MHC Class II mismatched, allogeneic graftsappeared comparable to autografts through POD-4. However, by POD-8, allallogeneic grafts demonstrated mild erythema, consistent with rejectionand were considered fully rejected by POD-10. No statisticallysignificant difference in the duration, quality of adherence, orcellular viability among the fresh, recently preserved, and long termpreserved skin grafts were observed. The cryopreserved materials were,statistically speaking, more alive than dead, and this finding wasempirically witnessed in vivo, as all 7-year grafts demonstratedadherence to the wound bed and prolonged survivability. Suchsurvivability would not have been exhibited by non-vital allografts.Without limiting the invention, it will be understood that the timeperiod for cryopreservation for the present invention may, for example,include any length of time up to about 7 years.

Materials and Methods:

The study was conducted in accordance an IACUC approved protocol(2005N000279, Amendment 69) at the Center for Transplantation Sciences,and in compliance with the U.S. Department of Agriculture's (USDA)Animal Welfare Act (9 CFR Parts 1, 2 and 3), the Guide for the Care andUse of Laboratory Animals, and all state, local laws and regulations.Study protocols, surgical procedures, and animal care guidelines wereindependently reviewed and monitored by a standing IACUC committee.

A total of eight swine were enrolled in this experiment, and all weremembers of the Sachs-NIH, inbred miniature swine colony. At the time ofsurgery, all swine were between 10 and 20-kg in total body weight andbetween 2 and 4 months of age. Immunosuppression regimen(s) were notadministered at any time during this experiment. Animals 24074 and 24075were assigned to Cohort 1 and represented a MHC-matched donor-recipientpair. Animals 24043 and 24070 were assigned to Cohort 2 and representeda mis-match of MHC Class II donor-recipient pair. Separately, for the invitro, MTT series of analyses, five, additional wild-type Göttingenminiature swine provided tissues for positive and negative controls.

Swine donors were anesthetized with I.M. 2 mg/kg telazol (tiletamine HCland zolazepam HCl, Zoetis Inc., Kalamazoo, Mich.) and brought to theoperating room for orotracheal intubation. Anesthesia was maintainedusing 2% isoflurane and oxygen. Skin surfaces were disinfected beforesurgery with chlorhexidine acetate (Nolvasan® Surgical Scrub, Fort DodgeAnimal Health, Fort Dodge, Iowa) and povidone-iodine, 10% (BetadineSolution, Purdue Products, L.P., Stamford, Conn.). The animals were thendraped, leaving the right side of the dorsum exposed. Split-thicknessskin grafts, measuring approximately 25 cm² (surface area) wereharvested between the scapula and inferior margin of the lowermost ribfrom each animal using an air-driven Zimmer dermatome (Medfix Solution,Inc., Tucson, Ariz.) with the depth set to 0.056-cm (0.022 inches).

Following skin graft harvest, grafts intended for cryopreservation andstorage for limited duration grafts underwent a standardizedinstitutional protocol and were maintained at −80° C. for 15 minutesprior to thawing. Long-term cryopreserved grafts had been continuouslystored at −80° C. for a period of more than 7 years. All grafts,previously sized to approximately 25 cm², were placed on a sterile nylonmesh backing for structural support and rolled for placement into athreaded seal cryovial under a laminar flow hood. Once all grafts wereprepared, approximately 5-mL of freeze media was added to the vial andsealed. The protocol required freeze media prepared by combining 15%dimethyl sulfoxide (DMSO) cryoprotective media (Lonza BioWhittaker) withfetal porcine serum (FPS) or donor serum (if FPS is unavailable) in a1:1 ratio, filtering (0.45 micron), and chilling to 4° C. prior to use.The vials were subsequently frozen in a controlled rate, phase freezerat a rate of 1° C. per minute to −40° C., then rapidly cooled to atemperature −80° C., at which they remained for 15 minutes for thosetest articles in the control group subjected to limited storageduration, or for a period of more than 7 years in the case of the thoseexperimental grafts in the test group exposed to extended duration ofcryopreservation. DMSO displaces intracellular fluid during the freezingprocess. Cryoprotective media, e.g., CryoStor is used in an amount ofabout 40-80%, or 50-70% based on maximum internal volume of the cryovial(10 ml) less the volume of the xenotransplantation product.

In order to thaw the grafts for surgical use, sealed vials were placedin 37° C. water baths for approximately 1 minute, at which point thevial was opened and the frozen graft was removed using steriletechnique. Subsequently, grafts underwent 3, 1-minute serial washes innormal saline with gentle agitation, in order to dilute andsystematically remove ambient, residual DMSO and prevent loss of cellviability. Grafts were then taken to the surgical field in normal salineat 25° C. for engraftment.

Two separate, but identical, surgical events were performed insuccession. The entire surgical plan included a total of four (n=4)donor-recipient swine, employing two animals per each of the twoexperimental cohorts (Cohort 1 and Cohort 2), paired intentionally basedon SLA-configurations as described previously. In total, four technicalcontrols and twelve (n=12) experimental grafts were engrafted andsubsequently observed.

Each animal received four deep-partial defects along the animal's rightdorsum, in a linear (caudal to cranial) orientation, ordered from 1 to4, respectively. Deep-partial wound defects were surgically introducedvia additional passes with the dermatome after the initial splitthickness graft harvest. The resulting wound beds were uniform, free ofvisible debris, and demonstrated independent, punctate bleeding. Thesedefects were interrupted, and not made in a single continuous pass withthe dermatome. Instead, care was given to create four, isolated butequivalent wounds with regards to overall size, depth, and anatomicallocation.

Following thawing, but prior to engraftment, all split-thickness skingrafts were fenestrated using a 15 (size) blade to prevent seroma orhematoma formation. Graft test articles were independently placed on theprepared wound bed and uniformly sutured in place using simpleinterrupted, 3-0 nylon sutures, applied in a graft-to-wound bed manner.Approximately 16 points of fixation were introduced per graft, spacedevenly around the graft, with the resulting knot located on the woundborder, not the graft article. This technique ensured that minimal, butadequate, residual tension was present and uniform, which is necessaryfor optimal graft-to-wound adherence, minimization of hematomas, andoptimal graft survivability.

At Wound Site 1 (most caudal), a split-thickness autograft was placed,serving as a technical control. This autograft test article washarvested during the wound bed creation, subsequently underwent the samefreeze-thaw process concomitantly with all experimental grafts, and washeld in an identical, cryopreserved state for the same duration as thecontrol grafts identified for a limited duration (15 minutes at −80°C.). At Wound Site 2, a split-thickness allograft from its respectivecohort pair-mate was sutured into place. This graft represented testarticles exposed to cryopreservation for a limited duration (15 minutesat −80° C.). At Wound Site 3, a split-thickness allograft from thewild-type donor, which represented a split-thickness graft, withidentical SLA matching as those at Wound Site 2 that had experienced“extended” storage in the cryopreserved state (more than 7 years at −80°C.). At Wound Site 4 (most cranial), a split-thickness allograft from agenetically engineered knockout donor, which represented asplit-thickness graft, with identical SLA matching as those grafts atWound Site 2, sourced from the genetically engineered donor animal, thathad also experienced “extended” exposure in the cryopreserved state(−80° C.) for more than 7 years.

Overlying pressure dressings, consisting of Xeroform petrolatum gauze(Medtronic), Telfa™ non-adhesive dressing (Covidien, Minneapolis,Minn.), and sterile gauze were maintained in place and dry withmultiple, overlapping sheets of Tegaderm™ (3M, St. Paul, Minn.).Recipients were then dressed with cotton jackets to reduce interferencewith the grafts. Graft dressings were removed on POD-2 and changed dailythereafter. Total postoperative follow up was 20 days. Animals weremonitored for signs of pain including vocalization, tachypnea, loss ofappetite, and changes in attitude, behavior, and mobility. Transdermalfentanyl patches were applied for post-operative analgesia. All sutureswere removed by POD-7.

To validate the assay method and establish boundary conditions specificto test articles of split thickness skin porcine skin, two independentassay series were performed on fresh (n=5, 5) and heat denatured samples(n=5, 5). The (geometric) average formazan produced on fresh samples was0.221±0.022-mg/mL and 0.300±0.035-mg/mL, respectively. In contrast, the(geometric) average formazan produced by heat-denatured samples was0.094±0.020-mg/mL and 0.105±0.009-mg/mL, respectively. These differenceswere statistically significant in both cases (p<0.05).

All four porcine recipients tolerated the surgical procedure andrecovered fully without incident. All sixteen (n=16) graftsre-vascularized without evidence of technical complication, anduniformly exhibited adherence to the underlying wound bed (i.e. “goodtake”). Over the course of the post-operative observational period, nografts were lost due to mechanical disturbance or exhibited any clinicalsigns of wound infection. All four (n=4) autografts at Wound Site 1healed permanently and were indistinguishable from surrounding tissuesat the study end-point, acting as a technical control for the skingrafting, cryopreservation and thawing technique.

In Cohort 1, all six (n=6) allogeneic grafts demonstrated equivalentadherence to the underlying wound bed and uniformly exhibited clinicalsigns consistent with vascularization and perfusion on postoperativedays (POD) 2 and 4. Notable, however, was the contrast (loss) of colorexhibited by the allografts that had been cryopreserved for an extendedduration. All four of these grafts appeared paler as compared to theautograft and allografts at Wound Site 2. This appearance fully resolvedin all grafts, in both Animals, by POD-6. All six (n=6) allograftsexhibited mild sloughing of the superficial epidermis by POD-8, butgrafts remained viable, adherent, and appeared otherwise healthy atinspection on POD-12. In Animal 24074, grafts at Wound Sites 2 and 3showed initial signs of necrosis, progressive erythema, and loss ofadherence by POD-14, and presented increasing signs of immune-mediatedrejection, until final rejection at POD-18. However, the allograft atWound Site 4 (most-cranial) did not similarly persist; instead, onPOD-14 this graft was significantly darker and exhibited signs ofcomplete necrosis and was clinically assessed to be fully rejected atthis time. The rapid loss of the graft 4, from viability at POD-12 tocomplete avulsion by POD-14, dissimilar and distinct from Wound Site 2and Wound Site 3, was notable. For grafts on Animal 24075, all graftswere rejected on POD-14.

In Cohort 2, animals presented similarly to those in Cohort 1 throughPOD-4, and equivalently to each other. Overall, clinical signs werecomparable in progression to the minor-mismatched grafts in Cohort 1,but at an accelerated pace. The grafts that had experienced extendedcryopreservation appeared paler at POD-2 and POD-4 than the grafts thathad not experienced cryopreservation, and all grafts showed increasedevidence of perfusion and vascularization by POD-6. By POD-8, all threeallogeneic grafts in Animal 24043, showed clear signs of rejection andwere considered fully rejected. In Animal 24070, all three allogeneicgrafts showed clear signs of rejection and were considered fullyrejected by POD-10. However, all allogeneic grafts survived at the samerate, irrespective of the genetics or length of storage.

With respect to grafts subjected to limited or extended durations ofcryopreservation, 100% of allograft comparators at Wound Sites 2 and 3(n=4 of 4) were identical with respect to clinical assessment ofduration of graft survival. Comparison of Wound Sites 2 and 4 werecoincident (n=3), with the exception of the allograft at Wound Site 4,Animal 24074, which survived until POD-14 (n=1), determined to beclinically and rejected four days prior to its counterparts.

Overall, histological assessments closely mirrored the clinicalassessments. Following surgery, all grafts, including autografts,exhibited early signs of acute inflammation during initial observationson POD-2 and 4, that later resolved with time. All allografts in Cohort2, as compared to those in Cohort 1, uniformly exhibited acceleratedprogression towards immune-mediated rejection.

Ultimately, all six (n=6) allogeneic grafts in Cohort 1, and threeallogeneic grafts (n=3) from Animal 24043 in Cohort 2, independentlydemonstrated histological and microscopic signs of rejection coterminouswith the independent gross clinical assessments. The single exceptionwere the three allografts engrafted on Animal 24070, where each graftreceived Banff scores of 4 (of 4) on POD-10, but were not deemedofficially rejected until POD-12, one assessment period (2 days) laterthan the corresponding clinical designation assigned at POD-10.

With respect to grafts subjected to limited or extended durations ofcryopreservation, 100% of allograft comparators at Wound Sites 2 and 3(n=4 of 4) were identical with respect to histological assessment ofduration of graft survival. Comparison of Wound Sites 2 and 4 werecoincident (n=3), with the exception of the allograft at Wound Site 4,Animal 24074, which survived 14 days post-operatively (n=1), determinedto be histologically rejected four days prior to its counterparts.

Neither the MTT nor the neutral red staining technique, as applied oneither testing occasion, were deemed effective for histological andmicroscopic evaluation, however the standard hemotoxylin and eosinstaining demonstrated observable tissue destruction of the heatdenatured specimens.

Overall, using a linear, mixed effect model with random intercept, themean survival of grafts at Wound Site 3 was 0.00 (95% CI: −1.10, 1.10days) less than allografts at Wound Site 2. The mean survival of graftsat Wound Site 4 was 2.00 (95% CI: 1.10, 3.10 days) less than allograftsat Wound Site 2. Histological assessment finds on average 0.5 days moresurvival than grafts assessed grossly, but this is not statisticallydistinguishable (p=0.28). Seven of the eight experimental grafts faredequivalently to their comparators. The in vivo experiments showed nostatistical difference between grafts subjected to short versuslong-term storage. With the exception of the graft at Wound Site 4 onAnimal 24074, which was assessed as fully rejected four days earlierthan its comparators, graft performance and survivability wereindistinguishable between the two groups.

As noted in previous publications, cryopreserved grafts appeared notablypaler during the early imbibition and vascularization periods. Thiscontrast was starkly evident for grafts at Wound Sites 3 and 4 in allanimals. Ultimately, grafts fully resolved and adhered to the underlyingwound bed to an equivalent degree.

Demonstrated viability was evidenced uniformly across the three,independent evaluation methods. The statistical analysis of theMTT-assay shows there was no significant difference betweencryopreserved and fresh specimens (FIG. 50A), but significantdifferences were observed between fresh and cryopreserved specimensversus heat-denatured ones (FIG. 50B). This suggests broadly that thecryopreserved materials were, statistically speaking, more alive thandead. This outcome is substantiated in the in vivo outcomes in which all7-year grafts demonstrated adherence to the wound bed and prolongedsurvivability, which would not be exhibited by non-vital grafts.

Regarding the MTT-reduction assays, substantial variability existedbetween absolute values resulting from such assays, from specimen tospecimen and from cohort-to-cohort. Indeed, absolute values of formazanproduction were actually higher than those obtained fromnon-cryopreserved samples; it is unlikely that freezing enhancedcellular activity.

Pig skin can be cryopreserved for years, e.g., 1, 3, 5, 7 or more yearsand retain cell viability and that the genetic modification, Gal-T-KO,did not impact metabolic stability when compared to wild type pig skinprocessed and stored using the same procedures.

Furthermore, the use of MHC-matched and Class II mismatcheddonor-recipient pairs in this model of allogeneic skin transplantationserved as internal controls to compare the effect of long termcryopreservation (7 years) on the survival of allogeneic skin grafts.The cell viability data after long term cryopreservation is supported bythe survival of the skin in vivo. This also demonstrated that thegenetic differences (wild type versus Gal-T-KO) of the grafts did notimpact the survival of the grafts.

The hypothesis was that graft take, and overall survival, would beinversely proportional to the length of storage duration. In otherwords, it was expected that the longer the graft had been frozen, theless likely it would survive and mimic the comparator grafts preservedfor shorter durations. Surprisingly, these studies revealed that theporcine tissue can be cryopreserved for significant durations, 7 yearsin the case of the present disclosure, and retain adequate cellviability. Moreover, the genetic modification (Gal-T-KO) did not impactmetabolic activity, when compared to wild type skin processedidentically. Lastly, the results confirm that the MTT-reduction assaycan reliably provide an accurate, useful diagnostic method, andapplicable to the assessment of porcine skin graft viability.

The promising results of this study indicate that it may be feasible tocryopreserve and store porcine skin for logistically relevant durations,and our findings are consistent with current industry practices and themulti-year “shelf life” guidance that the American Association forTissue Banks has established for human cadaveric tissues.

Further, these data indicate that scalable, clinically useful methods ofpreserving and storing porcine xenotransplantation products withadequate viability are disclosed, and that vital porcinexenotransplantation products that can be effectively stored anddistributed.

Example 4 Product Processing Generally

A xenotransplantation product of the present disclosure was processedaccording to the following procedures.

Personnel

The operator was dressed in sterile dress in accordance withinstitutional standards to maintain designated pathogen free conditions.The operator wore eye protection safety glasses for ultraviolet lightand lasers.

Preparation of Laminar Flow Hood and Product Processing

An ultraviolet laser lamp (Model #) was set up in a laminar flow hood.Each of the four corners of the lamp was placed on two container lidsthat were stacked on top of each other, i.e., four pairs of lids wereused to support the lamp. The distance from the lamp bulbs (2 bulb tubestotal) to the floor of the hood was approximately 1.5 inches. The entireinterior of the hood was sprayed with alcohol, e.g., ethanol orisopropanol. The lamp was turned on and the operator performed acalculation of time for desired exposure based on lamp specifications,number of bulbs, and distance between the bulbs and thexenotransplantation product.

The operator poured two baths (one chlorhexidine and one alcohol) intotwo separate bowls and placed the two bowls under the hood.

A package of new sterilized vials was placed under the hood. Vial capswere unscrewed and placed into the chlorhexidine bath. Each vial(without cap) was then turned upside down and plunged open ended intothe chlorhexidine bath, for one minute each and then set upright to airdry. Thereafter, the exterior of each vial was wiped with chlorhexidineand alcohol utilizing sterile gauze. The vial caps were removed from thechlorhexidine bath and placed on sterile gauze. The open ends of eachvial were plunged into alcohol bath for 1 minute each and then set asideto air dry.

A xenotransplantation product “#46 product” (5×15 cm) having a meshbacking prepared according to Example 2 was removed from its originalvial and the operator placed original vial into an empty bowl. Operatorplaced the #46 product on the paper side of an opened sterilizedinstrument package. The operator unrolled the #46 product and placed itunder the lamp for 2 minutes, then turned it over to the other side,removed the mesh backing, and put it under the lamp for 2 minutes onopposite side, while still on the same paper. The time period forexposing a given sample to the UV light can be varied based on thespecific biological agents or the types of biological agents to besterilized, e.g., as shown in the following Table 12:

TABLE 12 Type of UV-C Dosage (uW Sterilization Biological sec/cm²) for90% time Biological Agent Agent sterilization (sec)* Penicillium spp.Fungus 224,000 1800 Aspergillus flavus Fungus 34,900 300 Aspergillusniger Fungus 31,500 250 Yeast Fungus 4000 30 Influenza A Virus 1900 15HIV-1 Virus 28,000 220 Vaccinia Virus 1500 10 Escherichia coli Bacteria2000 20 Staphylococcus Bacteria 6600 50 aureus Bacillus subtilisBacteria 6800 50 Mycoplasma spp. Bacteria 8400 70 Pseudomonas Bacteria2200 20 aeruginosa *Using a UV-C intensity of 125 uW/cm²

Then the “#46 product was removed and cut in half. Each half was rolledby hand and placed into a new vial sterilized as explained above. Eachnew cap was placed on each new vial and screwed on securely. Each vialwas placed under the lamp and periodically rolled for desired evenexposure to light on the exterior of the vial. The vials were placedinside a glass jar that had an interior that had been previouslysterilized and the exterior was sterilized by the operator with alcoholand chlorhexidine, including threads and caps.

A similar process was performed for the following xenotransplantationproducts, except instead of being placed on sterile paper prior to entryunder the lamp, the mesh was not removed from the products and theproducts were placed under the lamp skin side up for 2 minutes, then theproducts were folded over so a first half of the bottom portion of eachproduct faced the lamp for 2 minutes, then the second half of eachproduct was folded over so that the other half of the bottom of eachproduct faced the lamp for 2 minutes. Some of the products were cut intosmaller sections and exposed to light, some for periods for longer than2 minutes, but never less than 2 minutes.

Products #40 (5×15 cm), #63 (10×15 cm), #69 (10×15 cm), and #25,underwent the above processes and products #69 and #25 were rolledexclusively using instruments and the operator did not directly handlethose products. As with #46, after operator securely screwed the cap oneach vial, each vial was placed under the lamp and rolled for evenexposure to light emitted from lamp. Vials were later removed from underthe lamp and wiped down with alcohol prior to being placed into glassjars.

Four glass jars were utilized to store each of the sets of vials. Priorto being handed to the operator, the assistant drenched the exteriors ofthe glass jars with alcohol via a spray bottle. The assistant handed theglass jars to the operator by holding the bottom of each jar and handingto operator outside of hood. After receiving the glass jars fromassistant, under the hood, the operator bathed the glass jar lids andplunged the open ends of the jars into alcohol and wiped the exterior ofthe jars with alcohol including threads of the jar.

The vials were wiped with alcohol utilizing gauze and placed inside eachglass jar with an instrument. The lids of the glass jars were thensecured and the jars were handed to the assistant. Frequently and on aperiodic basis throughout these processes the assistant sprayed theoperator's gloves and arms with alcohol.

Thereafter, the products were placed into the phase freezer at theconclusion of the procedures.

Example 5

In a human evaluation of a xenotransplantation product of the presentdisclosure for treatment of severe and extensive partial and fullthickness burns in a human patient, the following results were obtained:

The patient presented with a mixed depth, flame-induced burn injury,resulting in a 14% Total Body Surface Area (TBSA) defect to the(anatomic) right, upper torso—specifically, bordered: from the rightlateral axilla (superior border) to the sixth right lateral rib(inferior border) as shown in FIG. 51A.

The surgeon temporarily grafted part of the affected wound area withHuman Deceased Donor (HDD) allograft and the xenotransplant product ofthe present disclosure. The remaining regions of the wound area werecovered with a negative pressure wound therapy (NPWT). The patientreceived approximately 150 cm² of HDD allograft, meshed to a 1:1.5ratio, and 25 cm² of xenotransplant product of the present disclosuremeshed to a 1:1 ratio during surgery, which is specifically shown inFIG. 51B

Both temporary wound closure dressings were placed adjacently, but notin direct contact, and were secured with staples on the perimeter of thetissue(s), overlaid with NPWT.

Upon clinical visual inspection of the first wound dressing change onPOD-5, the HDD allograft and xenotransplant product of the presentdisclosure were both observed to be fully adherent to the underlyingwound bed and were indistinguishable as shown in FIG. 51C.

The patient experienced no adverse events and no serious adverse eventswere observed or reported.

In accordance with the regular clinical standard of care, both HDDallograft and the xenotransplant product of the present disclosure wereremoved at the first wound dressing change. Following mechanicalremoval, the underlying wound beds were equally perfused (with visiblepunctate bleeding) and otherwise appeared equivalent as shown in FIG.51D.

A POD-5 close-up image of the wound bed for the xenotransplant productof the present disclosure adjacent to wound bed for HDD allograft isshown in FIG. 51E.

On POD-5 following removal, per clinical standard of care, the entireaffected area received definitive wound closure via engraftment with aself (auto)graft (autologous split-thickness skin graft), obtained fromthe patient as shown in FIG. 51F.

Per protocol, blood samples for infectious disease, immunologicalresponse, and long-term evaluation were obtained, as well aspre-operative, peri-operative, and post-operative photographs.

On POD-14 (from the first operation), clinical observations at the wounddressing change demonstrated no discernible differences in the woundhealing rate or quality at any location as shown in FIG. 51G.

Per protocol, blood samples for infectious disease, immunologicalresponse, and long-term evaluation were obtained, as well aspre-operative, peri-operative, and post-operative photographs.

Testing for detection of PERV by quantitative RT-PCR was performed onbaseline blood samples (25 mL), first dressing change (21 mL), and twoweek blood samples (23 mL). The results were as follows:

PERV was not detected by qPCR in either RNA or DNA isolated from PBMCand RNA isolated from plasma. Evidence of porcine cells as determined byqPCR directed to the porcine mtCOII gene was not found in RNA isolatedfrom the PBMC.

Source Cq PERV pol Cq porcine mtCOII PERV Porcine cells DNA-PBMC <LOD<LOD Negative Negative RNA-PBMC <LOD <LOD Negative Negative RNA-plasma<LOD <LOD Negative Negative

Further, a study is conducted to assess the proliferative response ofhuman lymphocytes responder peripheral blood mononuclear cells (PBMC) inthe presence of mitomycin C treated porcine stimulator cells(alpha-galactosyltransferase knock out (KO) pig B173) over time. PBMCsamples were obtained from patients enrolled in Sponsor Study XT-001,both before and after the transplantation of porcine skin grafts. Theporcine skin grafts were obtained from genetically modifiedalpha-galactosyltransferase knock out (KO) pigs.

Patient PBMC samples were previously prepared by Ficoll gradientcentrifugation and cryopreserved. Whole blood from the skin donor pig(B173) was previously shipped to Xeno Diagnostics (XD) and PBMCsisolated by Ficoll gradient centrifugation and cryopreserved. The dayprior to setting up the MLR, samples were thawed at 37° C., washed, andrested overnight in 10% FBS/RPMI. Porcine PBMCs were mitomycin C treated(stimulators) and mixed with an equal number of test human PBMCs(responders). The MLR was incubated for seven days with BrdU added onday six. On day seven, a BrdU ELISA was performed and proliferationmeasured.

As shown in FIG. 52, PBMC obtained from skin graft Patient XT-001generated positive xenogeneic MLR PBMC mixed lymphocyte responses (MLR)when cocultured with alpha-Gal KO pig 173 PBMC (same source as skingraft). The xenogeneic proliferative responses were highest in culturesfrom sampling Days September 4 and September 19. In contrast, thexenogeneic proliferative response from sampling Day October 3 wasreduced and near autologous MLR response levels. Overall, the xenogeneicresponse with KO pig 173 in all time periods tested was less than thehuman IRB 11 allogeneic comparator.

Furthermore, a study is conducted to measure the levels of human plasmaanti-porcine IgM and IgG binding to porcine peripheral blood mononuclearcells (PBMCs) obtained from alpha-galactosyltransferase knock out (KO)pigs over time. Plasma samples are obtained from patients enrolled inSponsor Study XT-001, both before and after the transplantation ofporcine skin grafts. The porcine skin grafts were obtained fromgenetically modified alpha-galactosyltransferase knock out (KO) pigs.

In the study, the plasma samples were decomplemented in a 56° C. dryheat bath for 30 minutes. The samples were cooled and serially dilutedin FACS binding/washing media. The diluted plasma samples were thenincubated with KO porcine PBMCs followed by incubation with secondaryantibody (PE-Goat anti human IgG and FITC-Goat anti human IgM).Appropriate compensation, Fluorescence Minus One (FMO), and Limit ofBlank (LOB) controls were run in the same assay. Cells were acquired andanalyzed on an ACEA NovoCyte Flow Cytometer. Binding of anti-porcine IgMand IgG was assessed using Median Fluorescence Intensity (MFI) andrelative MFI obtained as follows: Relative MFI=Actual MFI value/LOB (MFIobtained using secondary antibody only in the absence of plasma).

The human plasma IgM and IgG binding was measured at four time pointsincluding pre-grafting and post grafting (Day 7, Day 16, Day 30). Allactual test samples at 1:2, and 1:10 dilutions showed MFI values higherthan LOB values. As shown in FIG. 53, an increase in anti-xenogeneic IgMand IgG levels was obtained above pre-existing levels on Day 16 and Day30 as shown by an increase in relative median fluorescence intensities.The average post-assay cell viability value determined by 7AAD was92.82%. Cells were only gated on ALIVE cells to determine IgM and IgGbinding to porcine PBMCs.

The invention claimed is:
 1. A biological system for generating andpreserving a repository of personalized, humanized transplantable cells,tissues, and organs for transplantation, wherein the biological systemis biologically active and metabolically active, the biological systemcomprising genetically reprogrammed cells, tissues, and organs in anon-human animal for transplantation into a human recipient, wherein thenon-human animal is a genetically reprogrammed swine forxenotransplantation of biologically active and metabolically activecells, tissue, and/or an organ isolated from the geneticallyreprogrammed swine, the genetically reprogrammed swine comprising anuclear genome that has been reprogrammed to replace a plurality ofnucleotides in a plurality of exon regions of a major histocompatibilitycomplex of a wild-type swine with a plurality of synthesized nucleotidesfrom a human captured reference sequence, and wherein cells of saidgenetically reprogrammed swine do not present one or more surface glycanepitopes selected from alpha-Gal, Neu5Gc, and SD^(a), and wherein genesencoding alpha-1,3 galactosyltransferase, cytidinemonophosphate-N-acetylneuraminic acid hydroxylase (CMAH), andβ1,4-N-acetylgalactosaminyltransferase are altered such that thegenetically reprogrammed swine lacks functional expression of surfaceglycan epitopes encoded by said genes, wherein the reprogrammed genomecomprises site-directed mutagenic substitutions of nucleotides at exonregions of: i) at least one of the wild-type swine's SLA-1, SLA-2, andSLA-3 with nucleotides from an orthologous exon region of HLA-A, HLA-B,and HLA-C, respectively, of the human captured reference sequence; andii) at least one the wild-type swine's SLA-6, SLA-7, and SLA-8 withnucleotides from an orthologous exon region of HLA-E, HLA-F, and HLA-G,respectively, of the human captured reference sequence; and iii) atleast one of the wild-type swine's SLA-DR and SLA-DQ with nucleotidesfrom an orthologous exon region of HLA-DR and HLA-DQ, respectively, ofthe human captured reference sequence, wherein intron regions of thewild-type swine's genome are not reprogrammed, and wherein thereprogrammed genome comprises at least one of A-C: A) wherein thereprogrammed swine nuclear genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type swine'sβ2-microglobulin with nucleotides from orthologous exons of a knownhuman β2-microglobulin from the human captured reference sequence; B)wherein the reprogrammed swine nuclear genome comprises a polynucleotidethat encodes a polypeptide that is a humanized beta 2 microglobulin(hB2M) polypeptide sequence that is at least 95% identical to the aminoacid sequence of beta 2 microglobulin glycoprotein expressed by thehuman captured reference genome; C) wherein the reprogrammed swinenuclear genome has been reprogrammed such that, at the swine'sendogenous β2-microglobulin locus, the nuclear genome has beenreprogrammed to comprise a nucleotide sequence encoding β2-microglobulinpolypeptide of the human recipient, wherein the reprogrammed swinenuclear genome has been reprogrammed such that the geneticallyreprogrammed swine lacks functional expression of the wild-type swine'sendogenous β2-microglobulin polypeptides, wherein said reprogrammingdoes not introduce any frameshifts or frame disruptions, wherein saidgenetically reprogrammed swine is free of at least the followingpathogens: (i) Ascaris species, cryptosporidium species, Echinococcus,Strongyloids sterocolis, and Toxoplasma gondii in fecal matter; (ii)Leptospira species, Mycoplasma hyopneumoniae, porcine reproductive andrespiratory syndrome virus (PRRSV), pseudorabies, transmissiblegastroenteritis virus (TGE)/Porcine Respiratory Coronavirus, andToxoplasma Gondii by determining antibody titers; (iii) PorcineInfluenza; (iv) the following bacterial pathogens as determined bybacterial culture: Bordetella bronchisceptica, Coagulase-positivestaphylococci, Coagulase-negative staphylococci, Livestock-associatedmethicillin resistant Staphylococcus aureus (LA MRSA), Microphyton andTrichophyton spp.; (v) Porcine cytomegalovirus; and (vi) Brucella suis,wherein said genetically reprogrammed swine is maintained according to abioburden-reducing procedure, said procedure comprising maintaining theswine in an isolated closed herd, wherein all other animals in theisolated closed herd are confirmed to be free of said pathogens, andwherein the swine is isolated from contact with any non-human animalsand animal housing facilities outside of the isolated closed herd. 2.The biological system of claim 1, wherein the genetically reprogrammedswine is non-transgenic.
 3. The biological system of claim 1, whereinthe wild-type swine genome comprises reprogrammed nucleotides atSLA-MIC-2 gene and at exon regions encoding SLA-3, SLA-6, SLA-7, SLA-8,SLA-DQ, CTLA-4, PD-L1, EPCR, TBM, TFPI, and beta-2-microglobulin usingthe human capture reference sequence, wherein the human cell, tissue, ororgan lacks functional expression of swine beta-2-microglobulin, SLA-1,SLA-2, and SLA-DR.
 4. The biological system of claim 3, wherein thewild-type swine genome comprises reprogrammed nucleotides at one or moreof a CTLA-4 promoter and a PD-L1 promoter, wherein the one or more ofthe CTLA-4 promoter and the PD-L1 promoter are reprogrammed to increaseexpression of one or both of reprogrammed CTLA-4 and reprogrammed PD-L1compared to the wild-type swine's endogenous expression of CTLA-4 andPD-L1.
 5. The biological system of claim 1, wherein a total number ofthe synthesized nucleotides is equal to a total number of the replacednucleotides, such that there is no net loss or net gain in number ofnucleotides after reprogramming the genome of the wild-type swine withthe synthesized nucleotides.
 6. The biological system of claim 1,wherein the nuclear genome is reprogrammed using scarless exchange ofthe exon regions, wherein the nuclear genome is reprogrammed withoutintroduction of any net insertions, deletions, truncations, or othergenetic alterations that would cause a disruption of protein expressionvia frame shift, nonsense, and missense mutations.
 7. The biologicalsystem of claim 1, wherein site-directed mutagenic substitutions aremade in porcine fetal fibroblast cell, a porcine zygote, a porcineInduced Pluripotent Stem Cells (IPSC), or a porcine germ-line cells. 8.The biological system of claim 1, wherein site-directed mutagenicsubstitutions are made in germ-line cells used to produce the non-humananimal.
 9. The biological system of claim 1, wherein the human capturedreference sequence is a human patient capture sequence, a humanpopulation-specific human capture sequence, or an allele-group-specifichuman capture sequence.
 10. The biological system of claim 1, whereinthe reprogrammed genome comprises site-directed mutagenic substitutionsof nucleotides at exon regions of the wild-type swine's MHC class Ichain-related 2 (MIC-2).
 11. The biological system of claim 1, whereinthe reprogrammed genome lacks functional expression of SLA-1, SLA-2,SLA-DR, or a combination thereof.
 12. The biological system of claim 1,wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type swine'sSLA-DQA from an orthologous exon region of a HLA-DQA1 captured referencesequence.
 13. The biological system of claim 1, wherein the reprogrammedgenome comprises site-directed mutagenic substitutions of nucleotides atexon regions of the wild-type swine's SLA-DQB from an orthologous exonregion of a HLA-DQB1 captured reference sequence.
 14. The biologicalsystem of claim 1, wherein the reprogrammed genome comprisessite-directed mutagenic substitutions of nucleotides at exon regions ofthe wild-type swine's SLA-DRA and SLA-DRB1 with nucleotides fromorthologous exon regions of HLA-DRA1 and HLA-DRB1 of the human capturedreference sequence, or wherein the reprogrammed genome lacks functionalexpression of SLA-DRA and SLA-DRB1.
 15. The biological system of claim14, wherein the site-directed mutagenic substitutions of nucleotides areat codons that are not conserved between the wild-type swine's nucleargenome and the known human sequence.
 16. The biological system of claim1, wherein the reprogrammed genome comprises site-directed mutagenicsubstitutions of nucleotides at exon regions of the wild-type swine'sSLA-DQA and SLA-DQB1 with nucleotides from orthologous exon regions ofHLA-DQA1 and HLA-DQB1 of the human captured reference sequence.
 17. Thebiological system of claim 1, wherein the reprogrammed genome comprisessite-directed mutagenic substitutions of nucleotides at exon regions ofSLA-3, SLA-6, SLA-7, SLA-8, and MIC-2.
 18. The biological system ofclaim 1, wherein the reprogrammed genome comprises site-directedmutagenic substitutions of nucleotides at exon regions of SLA-DQ andMIC-2.
 19. The biological system of claim 1, wherein the reprogrammedgenome comprises site-directed mutagenic substitutions of nucleotides atSLA-3, SLA-6, SLA-7, SLA-8, SLA-DQ, and MIC-2.
 20. The biological systemof claim 1, wherein said nuclear genome is reprogrammed to be homozygousat the reprogrammed exon regions, and wherein cells of said geneticallyreprogrammed swine have extracellular, phenotypic surface expression ofpolypeptides that are tolerogenic when the cells are transplanted intothe human recipient.